Compositions comprising DC-SIGN blockers and methods of using DC-SIGN blockers for preventing or treating diseases of a mammal, including viral infections

ABSTRACT

The present invention relates to methods and compositions for preventing or treating diseases of a mammal, including viral infections, wherein at least one symptom of the disease is mediated at least in part by the binding of an effector molecule to a DC-SIGN receptor present on cells of the mammal to be treated. The invention also provides methods of identifying compositions, wherein the compositions are useful for treating mammalian diseases, including viral infections, for which at least one symptom of the disease is mediated at least in part by the specific binding of an effector molecule to a DC-SIGN receptor present on the cells that express the DC-SIGN receptor, belonging to the mammal to be treated. The invention further relates to compositions and methods for targeting subject molecules to cells that express the DC-SIGN receptor.

[0001] Applicants claim the right to priority under 35 U.S.C. § 119(e)based on Provisional Patent Application Nos. 60/423,581, filed Nov. 5,2002, and 60/425,324, filed Nov. 12, 2002, both of which areincorporated herein by reference in their entirety.

BACKGROUND OF THE INVENTION

[0002] 1. Field of the Invention

[0003] This invention relates to methods and compositions for preventingor treating diseases of a mammal, wherein at least one symptom of thedisease is mediated at least in part by the binding of an effectormolecule to a DC-SIGN receptor of the mammal to be treated. The effectormolecule may be a molecule on a foreign organism. The foreign organismmay be a virus.

[0004] The invention also relates to compositions, and to methods ofidentifying compositions, wherein the compositions are useful fortreating mammalian diseases for which at least one symptom of thedisease is mediated at least in part by the binding of an effectormolecule to a DC-SIGN receptor of the mammal to be treated.

[0005] The invention further relates to compositions and methods fortargeting subject molecules to cells expressing DC-SIGN receptors, suchas dendritic cells. These compositions and methods are based ontargeting complexes, in which one or more subject molecules arecovalently attached to one or more DC-SIGN blockers and, by virtue ofbinding of one or more of the DC-SIGN blockers of the targeting complexto DC-SIGN, the subject molecule is targeted to cells expressing DC-SIGNreceptors.

[0006] 2. Description of the Related Art

[0007] Human Cytomegalovirus (CMV) is a double strand DNA virusbelonging to the Herpesviridae family and a ubiquitous pathogen inhumans. CMV interaction with its host is characterized by a primaryinfection followed by lifelong persistence in the host organism andviral reactivation episodes. CMV infection is asymptomatic in mostimmunocompetent individuals because of an efficient anti-viral immuneresponse. In contrast, CMV remains a major cause of morbidity andmortality in newborn and immunocompromised patients, namely inorgan-transplanted recipients or AIDS patients. In many cases, CMVdisease is characterized by a wide viral spread toward multiple organs(i.e. salivary glands, lung, kidney, gastrointestinal tract, liver,retina, CNS).

[0008] In vitro, a number of cell types are susceptible to CMV infectionwhen considering virus entry and viral immediate early gene expression.However, full replication of virus DNA and subsequent production ofinfectious virions is limited to permissive cells (i.e. fibroblasts,endothelial cells, the U373 MG astrocytoma cell line, etc.; see forreview Plachter et al., 1996). In fibroblasts (the prototypic cell typefor in vitro studies of CMV infection) CMV entry occurs in sequentialsteps involving several viral envelope (Env) glycoproteins. Initialattachment of virus to host cells is mediated through interactionbetween Env glycoproteins gB (CMV gB) and/or CMV gM with cell surfaceheparan sulfate proteoglycans (Compton et al., 1993; Kari and Gehrz,1992). Thereafter, binding of CMV gB with non-heparin cellular receptorsprobably allows more stable attachment of virus to cell surface (Boyleand Compton, 1998). Subsequent pH-independent fusion events betweenviral envelope and cell membrane are necessary for viral entry (Comptonet al., 1992; Milne et al., 1998). Cell proteins involved in CMVattachment and/or fusion have not been identified precisely although twocandidates have been proposed. The first one is annexin II whichinteracts with CMV gB (Pietropaolo and Compton, 1997). The second one isa 92.5 kDa protein binding to CMV gH (Baldwin et al., 2000). Fusionevents are followed by penetration of the capsid which is transported tothe nucleus. In some permissive cells, such as retinal pigmentepithelial cells, CMV can also penetrate into cells by a mechanism ofendocytosis (Bodaghi et al., 1999).

[0009] Recently, dendritic cells (DC), which are refractory to infectionby laboratory-adapted CMV strains, were shown to be permissive to CMVinfection and replication when infected with primary, clinical viralisolates (Riegler et al., 2000).

[0010] Dendritic cells are a diverse population of morphologicallysimilar cell types found in lymphoid or non-lymphoid tissues. Dendriticcells function as antigen-presenting cells that efficiently captureantigens in the peripheral tissues and process them to form MHC-peptidecomplexes. After antigen uptake, these immature dendritic cells acquirethe unique capacity to migrate from the periphery to the T cell areas ofthe secondary lymphoid organs. Dendritic cells convert antigens fromforeign cells and infectious microorganisms into short peptides that arebound to membrane proteins of the major histocompatibility complex(MHC). These MHC-peptide complexes are formed intracellularly, but areultimately presented on the plasma membrane where they serve as ligandsfor antigen-specific T cell receptors (TCR). In addition to TCR ligandformation, dendritic cells carry out many other functions, which allowthem to control immunity at several points (Steinman, 2000).

[0011] The mechanism of CMV entry into DC has not been investigated yet.It was recently shown that DC express a lectin called DC-SIGN(DC-Specific ICAM-Grabbing Nonintegrin). DC-SIGN, also called CD209, isa ligand for IntraCellular Adhesion Molecule-2 (ICAM-2) and ICAM-3(Geijtenbeek et al., 2000a; Geijtenbeek et al., 2000c) and is involvedin the attachment of Human Immunodeficiency Virus-1 (HIV-1) (Geijtenbeeket al., 2000b) and Ebola (Alvarez et al., 2002) to DC. DC-SIGN wasoriginally cloned from a placental cDNA library on the basis of itscapacity to bind to the surface subunit HIV-1 Env glycoprotein 120(HIV-1 gp120) (Curtis et al., 1992). DC-SIGN mediates HIV binding andinternalization into DC conferring to these cells the ability totransmit HIV to permissive CD4⁺ T cells independently from HIV-1replication (Geijtenbeek et al., 2000b). These findings suggest thatDC-SIGN efficiently captures HIV-1 at mucosal sites of inoculation andfacilitates its transport to sites of infection by using the migratoryability of DC towards lymphoid organs (Banchereau and Steinman, 1998). Ahomologue of DC-SIGN, DC-SIGNR, was recently identified on the surfaceof endothelial cells and shown to display the same HIV-1 binding andtrans-infection enhancement capacities shown by DC-SIGN (Bashirova etal., 2001; Pohlmann et al., 2001b). It has been suggested that theDC-SIGN lectin may act as a receptor for other glycan ligands present onother viral envelopes and on the cell walls of other microbes, or eventumor cells (Steinman, 2000). The putative role of DC-SIGN or DC-SIGNRin Herpesvirus attachment to DC or endothelial cells has never beenreported.

[0012] There exists a need in the art to develop methods andcompositions for modulating the specific binding of effector moleculesto the DC-SIGN receptor, for example on the dendritic cells of mammals.Such methods and compositions are needed, for example, to prevent andtreat diseases such as viral infections; for example CMV infections. Inthis regard, there is a need to identify cell proteins involved in viralattachment and/or fusion. Additionally, methods and compositions areneeded that allow the specific targeting of cells expressing DC-SIGNreceptor, such as dendritic cells or alveolar macrophages, to aid intherapy or diagnosis.

SUMMARY OF THE INVENTION

[0013] The inventors analyzed the mechanisms of CMV attachment to DC andthe role of DC-SIGN in this process. They demonstrated that CMV is ableto bind DC and DC-SIGN-expressing THP-1 cells through direct interactionof DC-SIGN with viral envelope CMV gB. Without in any way limiting theinvention, the inventors believe that this binding is involved in: (1)the transmission of DC-SIGN-bound infectious viral particles todifferent permissive cells and (2) an enhanced infection and CMVreplication in DC and DC-SIGN-expressing THP-1 cells.

[0014] Accordingly, this invention identifies DC-SIGN as a receptorinvolved in the binding of viruses other than HIV and Ebola virus todendritic cells. The invention further provides a number of novelmethods and compositions for treating diseases of mammals, includingviral infections.

[0015] A first object of the invention is to provide a method ofpreventing or treating a disease of a mammal, where at least one symptomof the disease is mediated at least in part by the binding of aneffector molecule to a DC-SIGN receptor of the mammal to be treated, andwhere the method comprises administering to the mammal an amount of aDC-SIGN modulator sufficient to substantially modulate the binding ofthe effector molecule to the DC-SIGN receptor to thereby prevent ortreat the disease.

[0016] Another object of the invention is to provide a method ofpreventing or treating a disease of a mammal, where at least one symptomof the disease is mediated at least in part by the binding of aneffector molecule to a DC-SIGN receptor of the mammal to be treated, andwhere the method comprises administering to the mammal an amount of aDC-SIGN blocker sufficient to substantially inhibit the binding of theeffector molecule to the DC-SIGN receptor to thereby prevent or treatthe disease.

[0017] In some embodiments the DC-SIGN blocker is a blocking derivativeof the effector molecule. In other embodiments the DC-SIGN blocker is anantibody.

[0018] Among embodiments of the invention where the DC-SIGN blocker isan antibody are included embodiments where the antibody specificallybinds DC-SIGN and embodiments where the antibody specifically binds theeffector molecule.

[0019] In some embodiments the DC-SIGN blocker is a mannosylatedmolecule that binds to a DC-SIGN receptor. The mannosylated molecule maybe mannan.

[0020] A further object of the invention is to provide a method ofpreventing or treating a viral infection of a mammal, where the viralinfection is mediated at least in part by the binding of a viraleffector molecule to a DC-SIGN receptor of the mammal to be treated,where the method comprises administering to the mammal an amount of aDC-SIGN modulator sufficient to substantially modulate the binding ofthe viral effector molecule to the DC-SIGN receptor to thereby preventor treat the viral infection.

[0021] Another object of the invention is to provide a preventing ortreating a viral infection of a mammal, where the viral infection ismediated at least in part by the binding of a viral effector molecule toa DC-SIGN receptor of the mammal to be treated, where the methodcomprises administering to the mammal an amount of a DC-SIGN blockersufficient to substantially inhibit the binding of the viral effectormolecule to the DC-SIGN receptor to thereby prevent or treat the viralinfection.

[0022] In some embodiments of the method the DC-SIGN blocker comprises abinding moiety of the viral effector molecule. In other embodiments theDC-SIGN blocker comprises a binding moiety of a viral envelopeglycoprotein. In other embodiments the DC-SIGN blocker is an antibody.The antibody may specifically bind DC-SIGN or specifically bind theviral effector molecule. In additional embodiments the DC-SIGN blockeris a mannosylated molecule that binds to a DC-SIGN receptor. Themannosylated molecule may be mannan.

[0023] Among embodiments of the invention in which the DC-SIGN blockeris an antibody are included embodiments in which the antibody is amonoclonal antibody; the mammal is a human and the antibody is amonoclonal antibody that is humanized; the antibody specifically bindsDC-SIGN; the monoclonal antibody is Mab 1B10.2.6; the antibodyspecifically binds the viral effector molecule; and the antibodyspecifically binds the binding moiety of the viral effector molecule.

[0024] In further embodiments of the method the viral effector moleculeis a molecular constituent of the viral envelope. In certain embodimentsthe molecular constituent of the viral envelope is an envelopeglycoprotein.

[0025] In additional embodiments of the method the DC-SIGN blockercomprises a binding moiety of the viral effector molecule. In someembodiments of the invention in which the viral effector molecule is amolecular constituent of the viral envelope the DC-SIGN blocker that isused comprises a binding moiety of the envelope glycoprotein.

[0026] In a preferred aspect of the invention the viral infection is aCMV infection and the viral effector molecule is a CMV effectormolecule. In a further preferred aspect the mammal is a human. In someembodiments the CMV effector molecule is a molecular constituent of theCMV envelope. In further embodiments the molecular constituent of theCMV envelope is a CMV envelope glycoprotein. In yet further embodimentsthe CMV envelope glycoprotein is CMV envelope glycoprotein B.

[0027] Included among embodiments of the invention in which the viralinfection is a CMV infection and the viral effector molecule is a CMVeffector molecule are embodiments where the DC-SIGN blocker comprises abinding moiety of the CMV effector molecule; the DC-SIGN blockercomprises a binding moiety of the CMV envelope glycoprotein B; theDC-SIGN blocker is a recombinantly produced protein; and the DC-SIGNblocker is an antibody. Among embodiments where the DC-SIGN blocker isan antibody are embodiments where the antibody is a monoclonal antibody;the mammal is a human and the monoclonal antibody is humanized; theantibody specifically binds DC-SIGN; the monoclonal antibody is Mab1B10.2.6; and the antibody specifically binds the CMV effector molecule.Among embodiments where the antibody specifically binds the CMV effectormolecule are embodiments where the CMV effector molecule is CMV envelopeglycoprotein B.

[0028] In a further aspect the invention provides a method of preventingor treating an Ebola, HIV or SIV infection of a human or a simian, wherethe method comprises administering to the human or simian an amount of aDC-SIGN modulator sufficient to substantially modulate the binding ofHIV or SIV to the DC-SIGN receptor present on dendritic cells of thehuman or simian to thereby prevent or treat the HIV or SIV infection.

[0029] In another aspect the invention provides a method of preventingor treating an Ebola, HIV or SIV infection of a human or a simian, wherethe method comprises administering to the human or simian an amount of aDC-SIGN blocker sufficient to substantially inhibit the binding of HIVor SIV to the DC-SIGN receptor present on dendritic cells of the humanor simian to thereby prevent or treat the HIV or SIV infection. In apreferred embodiment the DC-SIGN blocker comprises a binding moiety ofthe CMV envelope glycoprotein B. In another preferred embodiment an HIVinfection of a human is prevented or treated.

[0030] In a further aspect the invention provides a method of preventingor treating an Ebola, HIV or SIV infection of a human or a simian, wherethe method comprises administering to the human or simian an amount of aDC-SIGN modulator sufficient to substantially modulate the binding ofHIV or SIV to the DC-SIGN receptor present on dendritic cells of thehuman or simian to thereby prevent or treat the HIV or SIV infection.

[0031] In another aspect the invention provides a method of preventingor treating an Ebola, HIV or SIV infection of a human or a simian, wherethe method comprises administering to the human or simian an amount of aDC-SIGN blocker sufficient to substantially inhibit the binding of HIVor SIV to the DC-SIGN receptor present on dendritic cells of the humanor simian to thereby prevent or treat the HIV or SIV infection. In apreferred embodiment the DC-SIGN blocker comprises a binding moiety ofthe CMV envelope glycoprotein B. In another preferred embodiment an HIVinfection of a human is prevented or treated.

[0032] In a further aspect the invention provides a pharmaceuticalcomposition comprising:

[0033] a) A DC-SIGN modulator, and

[0034] b) at least one pharmaceutically acceptable excipient;

[0035] wherein the DC-SIGN blocker is present in the composition at anachievable therapeutic concentration.

[0036] In a further aspect the invention provides a pharmaceuticalcomposition comprising:

[0037] c) A DC-SIGN blocker, and

[0038] d) at least one pharmaceutically acceptable excipient;

[0039] wherein the DC-SIGN blocker is present in the composition at anachievable therapeutic concentration.

[0040] In some embodiments of the pharmaceutical composition the DC-SIGNblocker is a derivative of a viral effector molecule. In one embodimentDC-SIGN blocker comprises the binding moiety of a CMV effector molecule.In another embodiment the CMV effector molecule is CMV envelopeglycoprotein B.

[0041] In other embodiments of the pharmaceutical composition theDC-SIGN blocker is an antibody. Embodiments where the DC-SIGN blocker isan antibody include embodiments where the antibody is a monoclonalantibody; the monoclonal antibody is humanized; the antibodyspecifically binds DC-SIGN; the monoclonal antibody is Mab 1B10.2.6; theantibody specifically binds the viral effector molecule; or the antibodyspecifically binds the binding moiety of the viral effector molecule.

[0042] In a further aspect the invention provides a method ofidentifying a DC-SIGN modulator, wherein the method comprises:

[0043] a) determining a baseline binding value by:

[0044] i. providing cultured cells comprising a DC-SIGN receptor;

[0045] ii. exposing the cultured cells to a marked viral effectormolecule binding moiety for a period of time sufficient to allow bindingequilibrium to be reached; and

[0046] iii. determining the extent of binding of the marked viraleffector molecule binding moiety to the cultured cells to therebydetermine a baseline binding value;

[0047] b) determining a test substance binding value by:

[0048] i. providing cultured cells comprising a DC-SIGN receptor;

[0049] ii. exposing the cultured cells to a marked viral effectormolecule binding moiety in the presence of a test substance for a periodof time sufficient to allow binding equilibrium to be reached; and

[0050] iii. determining the extent of binding of the marked viraleffector molecule binding moiety to the cultured cells to therebydetermine a test substance binding value; and

[0051] c) determining a test substance binding modulation value for thetest substance by dividing the test substance binding value by thebaseline binding value,

[0052] wherein a test substance binding inhibition value representing anabout 95% modulation of binding of the viral effector molecule todendritic cells by the test substance, indicates that the test substanceis a substance that substantially modulates the binding of a viraleffector molecule to the DC-SIGN receptor.

[0053] In a preferred aspect the invention provides a method ofidentifying a DC-SIGN blocker, wherein the method comprises:

[0054] a) determining a baseline binding value by:

[0055] i. providing cultured cells comprising a DC-SIGN receptor;

[0056] ii. exposing the cultured cells to a marked viral effectormolecule binding moiety for a period of time sufficient to allow bindingequilibrium to be reached; and

[0057] iii. determining the extent of binding of the marked viraleffector molecule binding moiety to the cultured cells to therebydetermine a baseline binding value;

[0058] b) determining a test substance binding value by:

[0059] i. providing cultured cells comprising a DC-SIGN receptor;

[0060] ii. exposing the cultured cells to a marked viral effectormolecule binding moiety in the presence of a test substance for a periodof time sufficient to allow binding equilibrium to be reached; and

[0061] iii. determining the extent of binding of the marked viraleffector molecule binding moiety to the cultured cells to therebydetermine a test substance binding value; and

[0062] c) determining a test substance binding inhibition value for thetest substance by dividing the test substance binding value by thebaseline binding value,

[0063] wherein a test substance binding inhibition value representing anabout 95% inhibition of binding of the viral effector molecule todendritic cells by the test substance, indicates that the test substanceis a substance that substantially inhibits the binding of a viraleffector molecule to the DC-SIGN receptor.

[0064] The method of identifying a DC-SIGN blocker includes embodimentswhere the cultured cells are DC; the cultured cells are THP-1 cells; theviral effector molecule is a CMV effector molecule; and the CMV effectormolecule is CMV envelope glycoprotein B.

[0065] In a further aspect the invention provides an isolated DC-SIGNblocker identified by the above method of identifying a DC-SIGN blocker.

[0066] In another aspect the invention provides a method of targeting asubject molecule to a cell expressing a DC-SIGN receptor by exposing thecell to a targeting complex, where the targeting complex comprises asubject molecule and a DC-SIGN blocker, and where the exposure is underconditions which allow the DC-SIGN blocker to bind to DC-SIGN on thecell expressing the DC-SIGN receptor, thereby targeting the subjectmolecule to the cell expressing a DC-SIGN receptor.

[0067] The method of targeting a subject molecule to a cell expressing aDC-SIGN receptor includes embodiments where the DC-SIGN blocker is anantibody; the DC-SIGN blocker is a monoclonal antibody; the subjectmolecule is a protein; the subject molecule is an antibody; the subjectmolecule is labeled; the exposure occurs in vivo; and the exposureoccurs in vitro.

[0068] In a further aspect the invention provides an isolated antibody,wherein the isolated antibody specifically binds DC-SIGN. In oneembodiment, the antibody is a DC-SIGN modulator. In a preferredembodiment, the antibody is a DC-SIGN blocker. In a further embodiment,the invention provides an isolated monoclonal antibody, wherein theisolated monoclonal antibody specifically binds DC-SIGN. In oneembodiment the monoclonal antibody is a DC-SIGN modulator. In apreferred embodiment, the monoclonal antibody is a DC-SIGN blocker. Inthe further preferred embodiment, the isolated monoclonal antibody isMab 1B10.2.6, produced by hybridoma 1B10.2.6, deposited at the C.N.C.M.on Nov. 7, 2002, under the accession number I-2951.

BRIEF DESCRIPTION OF THE DRAWINGS

[0069] The invention will be more fully described with reference to thedrawings in which:

[0070]FIG. 1 shows that DC-SIGN-expressing cells bind CMV on theirsurface. (A) Detection of DC-SIGN. Anti-DC-SIGN 1B10.2.6 mAb (boldline). Irrelevant isotypic control mAb (dotted line). Mean fluorescenceintensity (MFI) values are indicated. (B) Binding of CMV AD169 strain tocells expressing or not DC-SIGN was revealed by an anti-envelopeglycoprotein B (CMV gB) mAb. Incubation in low pH buffer (200 PFU/cell)prior to staining abrogates detection of CMV gB.

[0071]FIG. 2 depicts DC-SIGN-dependent trans-infection of CMV permissivecells. (A) MD-DC, (B) DC-SIGN⁺ vs parental THP-1 cells or (C) parentalvs DC-SIGN⁺ HeLa cells were pre-treated with EGTA (5 mM), mannan (20, 1,0.05 μg/ml), anti-DC-SIGN 1B10.2.6 mAb (20, 1, 0.05 μg/ml) or anisotypic control (20, 1, 0.05 μg/ml) prior to incubation with ADGFPstrain (1 PFU/cell). After removal of unbound virus and competitors,ADGFP-challenged cells were co-cultured for 3 days with MRC-5 cells.Infection of MRC-5 was assessed by counting the number of GFP-expressingcells by flow cytometry.

[0072]FIG. 3 shows that DC-SIGN enhances CMV trans-infection of MRC-5cells and retains long term infectious virus. (A) MD-DC, (B) parental orDC-SIGN⁺ THP-1 cells, were incubated with ADGFP in the absence or in thepresence either of anti-DC-SIGN 1B10.2.6 mAb or an isotypic, controlmAB. Thereafter, cells were co-cultured with reporter MRC-5 cells. (C)DC-SIGN⁺ and parental THP-1 cells were incubated (4 hours) with ADGFP(MOI=1) and washed thereafter. At days 0, 2, 4, 6, 8 after pulse,ADGFP-challenged cells were co-cultured with reporter MRC-5 cells for 3days. In (A), (B) and (C), MRC-5 cells were incubated with thecorresponding amount of cell-free virus to monitor kinetic and extent ofinfection. Values represent the percentage of MRC-5 cells expressingGFP.

[0073]FIG. 4 Shows that DC-SIGN mediates CMV transmission to differentpermissive cells but does not allow transmission of HSV-1 and VZV. (A)and (B), DC-SIGN⁺ THP-1 cells were exposed either to anti-DC-SIGN1B10.2.6 mAb or an isotypic control mAb prior to infection with ADGFP.ADGFP-pulsed DC-SIGN⁺ THP-1 cells were co-cultured for 24 hours eitherwith MRC-5 (A) or U373 MG cells (B). GFP- (grey bars) or CMVIEA/EA-expressing cells (open bars) were counted to evaluate CMVinfection. (C) DC-SIGN⁺ THP-1 cells incubated with anti-DC-SIGN 1B10.2.6mAb (open bars), an isotypic control mAb (dashed bars) or left untreated(grey bars) were exposed for 1 hr to TB40/E, AD169 or Towne CMVisolates, HSV-1 or VZV. After removal of unbound virus and mAb, cellswere co-cultured with MRC-5 reporter cell line for 5 days. (D) MRC-5cells were exposed for 5 days to identical amount of cell-free virusesas used in C to infect DC-SIGN⁺ THP-1 cells. IEA/EA-expressing cellswere detected by immunocytochemistry using specific antibodies for eachherpesvirus.

[0074]FIG. 5 Shows that the DC-SIGN cytoplasmic domain is required forCMV transmission. (A) Cell surface expression of wt or mutated DC-SIGN(Δ35 and Δ20) receptors analyzed by immunostaining (1B10.2.6 mAb) andflow cytometry. (B) CMV-binding capacity of THP-1 expressing wt ortruncated DC-SIGN revealed by anti-CMV gB mAb. (C) Parental, or DC-SIGN⁺cells were incubated, either at 4° C. or at 37° C. for 2 hours, withADGFP CMV (MOI=0.1) and co-cultured with MRC-5 cells for 3 days.Infection was assessed by estimating the number of GFP-expressing cells.

[0075]FIG. 6 Shows that DC-SIGN expression renders susceptible cellspermissive to CMV infection. A) Cells were pre-treated for 30 min withmedium (grey bars), anti-DC-SIGN 1B10.2.6 mAb (open bars) or an isotypiccontrol mAb (dashed bars) and thereafter incubated with ADGFP strain (1PFU/cell). HEK 293T were transiently transfected either with a DC-SIGNcDNA plasmid or a control plasmid (pcDNA3.1). (B) HEK 293T cells weretransiently transduced with DC-SIGN or DC-SIGNR cDNAs and incubated withADGFP. In (A) and (B), GFP-expressing cells were quantified by flowcytometry at 3 day after infection. (C) THP-1, DC-SIGN⁺ THP-1 cells orMD-DC were pre-treated as described in (A) (same symbols) and infectedwith TB40/E CMV (MOI=1) for 3 days. CMV infection was assessed byimmunostaining with specific CMV IEA/EA mAb. Total number of CMVIEA/EA-positive cells on the slide was determined by extrapolating thenumber of positive cells contained in the optical field of themicroscope (×10 objective). (D) MD-DC, MRC-5, parental and DC-SIGN⁺THP-1 cells were pre-treated and infected as described in (C).Non-internalized viral particles were removed by short incubation in alow pH buffer. At day 14 after infection virions released in culturesupernatants, were titrated on MRC-5 cells by plaque assays. Figures ontop of histograms indicate infection fold amplification which resultfrom dividing the absolute number of CMV particles collected insupernatants by the absolute number of CMV particles used to infectcells (20,000 PFU).

[0076]FIG. 7 depicts data that identify CMV gB as a viral ligand forDC-SIGN and characterization of the DC-SIGN/CMV gB interaction by SPR.(A) Binding of CMV gB to DC-SIGN. DC-SIGN⁺ THP-1 cells or MD-DC werepre-treated with medium (black closed circles), anti-DC-SIGN 1B10.2.6mAb (open circles) or an isotypic, control mAb (grey closed circles),and thereafter incubated with biotinylated CMV gB. Cell-bound CMV gB wasrevealed by PE-labelled streptavidin. Incubation of parental THP-1 cellswith biotinylated CMV gB is also shown (black closed triangles). (B)Competition assay of biotinylated HIV-1 gp120 binding to DC-SIGN.Parental (upper panel) or DC-SIGN⁺ THP-1 cells (all other panels) wereincubated with 2 Pg/mL of biotinylated HIV-1 gp120. DC-SIGN⁺ THP-1 cellswere left untreated or pre-incubated with potential competitors(unlabelled HIV-1 gp120, mannan, anti-DC-SIGN 1B10.2.6 mAb, controlisotypic mAb, or envelope glycoproteins from CMV gB, HSV-1 gB and gD orVZV gE and gB) before incubation with biotinylated HIV-1 gp120. MFI ofbiotinylated HIV-1 gp120 staining is indicated in the upper-right cornerof histograms. In each panel, control staining (dotted line),biotinylated HIV-1 gp120 labelling in the absence of competitor(gray-filled profile) or after pre-incubation with competitors(black-filled histogram), are shown. (C) Binding of CMV gB to DC-SIGNR.HEK 293T cells were transiently transfected either with a controlplasmid or plasmids encoding DC-SIGN or DC-SIGNR cDNAs. Transfectedcells were incubated with increasing concentrations of biotinylated-CMVgB (dashed bars), biotinylated-HIV-1 gp120 (black bars) orbiotinylated-BSA (open bars). Binding of biotinylated proteins wasrevealed by PE-conjugated streptavidin and analyzed by flow cytometry.Values are represented as MFI. (D) SPR analysis of DC-SIGN/CMV gBinteraction. The recombinant soluble CRD of DC-SIGN at (from bottom totop) 0.13, 0.21, 0.36, 0.6 or 1 μM was injected over surfaces coatedwith HIV-1 gp120 (left panel), CMV gB (middle panel) or HSV-1 gB (rightpanel) to analyze the association phase, after which running, bufferalone was injected to analyze the dissociation phase. Binding responses(Response Unit, RU) are reported as a function of time. Dissociationconstants (Kd) are indicated for left and middle panels.

DETAILED DESCRIPTION OF THE INVENTION

[0077] This invention relates to a method of preventing or treating adisease of a mammal, where at least one symptom of the disease ismediated at least in part by the binding of an effector molecule to aDC-SIGN receptor of the mammal to be treated. The method comprisesadministering to the mammal an amount of a DC-SIGN blocker sufficient tosubstantially inhibit the binding of the effector molecule to theDC-SIGN receptor to thereby prevent or treat the disease.

[0078] “Mammal” for purposes of the invention refers to any animalclassified within the class mammalia. Nonlimiting examples of mammalsinclude: humans and simians; pet animals, such as dogs, cats, ferrets,and guinea pigs; farm animals, such as pigs, cows, horses, sheep, goats,and llamas; and zoo animals, such as bears, zebras, elephants, and waterbuffalo. The mammal is preferably human.

[0079] As used herein a “disease” is any pathological condition of amammal, which results, for example, from infection, genetic defect, orexposure to a substance in the environment. The methods and compositionsof the invention are useful for preventing or treating diseases that arecharacterized in that at least one symptom of the disease is mediated atleast in part by the binding of an effector molecule to the DC-SIGNreceptor present on cells such as dendritic cells or alveolarmacrophages of the mammal. Specific examples of such diseases includeviral infection. A specific examples of viral infections that can betreated by the method is CMV infection of a human.

[0080] In the case of humans “DC-SIGN receptor” refers generically toDC-SIGN (described in Curtis et al., 1992) and/or DC-SIGNR (described inPohlmann et al., 2001.), and/or a homologue of DC-SIGN or DC-SIGNR. Oneof skill in the art will recognize that there may be some situations inwhich use of one or the other of these forms of DC-SIGN receptor ispreferable or even necessary. One of skill in the art will recognizethat human DC-SIGN protein can be obtained from many sources. Forexample, human DC-SIGN can be purified from human dendritic cells whichare obtained from an in vivo source, such as human blood, or purifiedfrom an in vitro source, such as human dendritic cells produced intissue culture from human dendritic cell precursor cells. It is alsopossible to express human DC-SIGN using a recombinant system, usingeither cultured dendritic cell as a host or a suitable heterologous celltype, such as COS-7 or HeLa cells, or bacteria such as E. coli.

[0081] In the case of nonhuman mammals, “DC-SIGN receptor” refers tohomologues of a human DC-SIGN receptor. One of skill in the art willrecognize that such proteins may be identified in any of a number ofdifferent ways. These include expression cloning, polymerase chainreaction using degenerate oligonucleotide primers, and low stringencyscreening of a bacterial or bacteriophage library.

[0082] Dendritic cells are a diverse population of morphologicallysimilar cell types found in lymphoid or non-lymphoid tissues. Dendriticcells function as antigen-presenting cells that efficiently captureantigens in the peripheral tissues and process them to form MHC-peptidecomplexes. Dendritic cells are also involved in the early activation ofnon-MHC-restricted γδ and CDI-restricted T cells specific for variousmycobacterial glycolipids, including CAM (Kaufmann, 2001 and Moody, etal., 2000). After antigen uptake, these immature dendritic cells acquirethe unique capacity to migrate from the periphery to the T cell areas ofthe secondary lymphoid organs. Dendritic cells convert antigens fromforeign cells and infectious microorganisms into short peptides that arebound to membrane proteins of the major histocompatibility complex(MHC). These MHC-peptide complexes are formed intracellularly but areultimately presented on the plasma membrane where they serve as ligandsfor antigen-specific T cell receptors (TCR). In addition to TCR ligandformation, dendritic cells carry out many other functions, which allowthem to control immunity at several points (Steinman, 2000).

[0083] Alveolar macrophages and dendritic cells are examples of cellsexpressing a DC-SIGN receptor. Endothelial cells are an example of cellsexpressing DC-SIGNR.

[0084] One of skill in the art will appreciate that dendritic cells maybe obtained from an in vivo source, such as the blood of a mammal, orgrown in vitro, by culturing dendritic cell precursor cells underappropriate conditions. Dendritic cell precursor cells include monocytesprepared according to Example 3.

[0085] An “effector molecule” is any molecule that specifically binds tothe DC-SIGN receptor present on cells of a mammal, such as the dendriticcells or the alveolar macrophages of a mammal, and thereby mediates asymptom that is associated with a disease of that mammal. Examples ofeffector molecules are ligands present on viruses that bind to receptorson cells of a mammal and thereby facilitate the entry of the virus intoa cell of the mammal. In cases where the effector molecules are ligandspresent on viruses the effector molecules can be referred to as “viraleffector molecules.” Examples of this type of ligand include gp120 ofHIV and envelope glycoprotein B of CMV, which bind with the DC-SIGNreceptor present on cells such as dendritic cells or alveolarmacrophages of a human to facilitate, in the case of CMV, thetransmission of DC-SIGN-bound infectious viral particles to differentpermissive cells and an enhanced infection and CMV replication in DC andDC-SIGN-expressing THP-1 cells. CMV envelope glycoprotein B is thus a“CMV effector molecule.” Other types of effector molecules are ligandsthat are endogenous to the mammal. This type of ligand includes bothligands that are bound to the surface of other cells of the mammal andsoluble ligands, which may be localized to the extracellular space of aparticular tissue or circulating systemically.

[0086] A “symptom” is any pathological manifestation of the disease tobe treated. A symptom is caused at least in part by the binding of aneffector molecule to the DC-SIGN receptor present on the dendritic cellsof the mammal to be treated if a modulation (a reduction or an increase)in the binding of the effector molecule to the DC-SIGN receptor causes adeterminable reduction in the occurrence or severity of the symptom, orboth. In a preferred embodiment of the invention the symptom is nolonger present or is prevented from occurring following the reduction inthe binding of the effector molecule to the DC-SIGN receptor.

[0087] An effector molecule is said to “specifically bind” to theDC-SIGN receptor present on cells such as the dendritic cells or thealveolar macrophages of the mammal to be treated if such binding is notcompetitively inhibited by the presence of unrelated molecules (e.g.,fetal calf serum), but is inhibited by antibodies to DC-SIGN (e.g.,1B10.2.6) and/or additional effector molecule.

[0088] An example of an effector molecule that specifically binds to theDC-SIGN receptor present on cells such as the dendritic cells or thealveolar macrophages of a mammal to be treated is CMV envelopeglycoprotein B.

[0089] One of skill in the art will appreciate that these assays may beused to identify other effector molecules that specifically bind to theDC-SIGN receptor present on cells such as dendritic cells of a mammal tobe treated. It will also be clear to one of skill in the art that otherequivalent assays may be substituted for those specifically disclosed inthe Examples.

[0090] Once an effector molecule is known to specifically bind to theDC-SIGN receptor the binding of the effector molecule to DC-SIGN can bereferred to simply as “binding.” It will be understood by one of skillin the art that such binding is specific. In this regard, the“modulation” of binding may be discussed. Modulation can include“inhibition” or “enhancement”.

[0091] “Modulation” means the act of regulating. It includes the act ofinducing variations of a property of a molecule. In the context of thepresent invention, “modulation” means the act of regulating and varyingthe binding of effector molecules to their receptors. This modulationmay serve to either inhibit or enhance binding, or to impose otherregulatory controls.

[0092] In the context of the present invention “inhibition” of bindingmeans a reduction in the total amount of effector molecule that binds toDC-SIGN over a fixed period of time. Inhibition of binding of theeffector molecule is achieved by providing a DC-SIGN blocker. A “DC-SIGNblocker” is any molecule that substantially inhibits the binding of agiven effector molecule at a concentration at which the effectormolecule specifically binds to DC-SIGN. In a preferred embodiment, theDC-SIGN blocker used is a monoclonal antibody that specifically bindsDC-SIGN. In another preferred embodiment the DC-SIGN blocker usedcomprises a binding moiety of the CMV envelope glycoprotein B.

[0093] In the context of the present invention “enhancement” of bindingmeans an increase in the total amount of effector molecule that binds toDC-SIGN over a fixed period of time. Enhancement of binding of theeffector molecule is achieved by providing a DC-SIGN enhancer. A“DC-SIGN enhancer” is any molecule that substantially enhances thebinding of a given effector molecule at a concentration at which theeffector molecule specifically binds to DC-SIGN.

[0094] A “binding moiety” is that portion of a molecule thatsubstantially retains the ability to bind to a second molecule whenother portions of the molecule are removed or modified or when thebinding moiety is placed into a heterologous context. For example, inthe case of an effector molecule as defined herein, a binding moiety ofthe effector molecule can be defined. A binding moiety of an effectormolecule is that portion of the effector molecule that substantiallyretains the ability to bind to DC-SIGN when other portions of themolecule are removed or modified or when the binding moiety is placedinto a heterologous context. In this context, “substantially retains”can be defined by one of skill based on the specific properties of thebinding moiety that are sought.

[0095] “Substantially inhibit” means greater than 80% inhibition,greater than 90% inhibition, greater than 95% inhibition, or greaterthan 99% inhibition. In a preferred embodiment of the present inventionabout 90% binding inhibition is obtained.

[0096] “Inhibition” is measured by comparing the extent of effectormolecule binding to DC-SIGN in the presence of a DC-SIGN blocker withthe extent of effector molecule binding to DC-SIGN in the absence of aDC-SIGN blocker. The ratio of extent of binding in the presence of theDC-SIGN blocker compared to the extent of binding in the absence of theDC-SING blocker is then determined. The percent inhibition is then theproportional reduction in the amount of binding. For example, a ratio of0.1 represents a 90% reduction in binding.

[0097] The term “treat,” “treating” or “treatment” refers to theadministration of therapy to an individual who already manifests atleast one symptom of a disease. Such an individual includes anindividual who is diagnosed as having a known disease.

[0098] The term “prevent,” “preventing” and “prevent” refers to theadministration of therapy on a prophylactic or preventative basis to anindividual who may ultimately acquire the disease but who has not yetdone so (i.e., those needing preventative measures). Such individualsmay be identified on the basis of risk factors that are known tocorrelate with the subsequent occurrence of the disease.

[0099] The term “therapeutic benefit” refers to an improvement of atleast one symptom of a disease, a slowing of the progression of adisease, as manifested by a slowing in the increase in severity of atleast one symptom of a disease, or a cessation in the progression of atleast one symptom of a disease. The therapeutic benefit is determined bycomparing a symptom of a disease before and after a DC-SIGN blocker isadministered.

[0100] The term “antibody” refers to any antibody that can be made byany technique known in the art. Suitable antibodies are obtained byimmunizing a host animal with peptides comprising all or a portion ofthe target protein. Suitable host animals include mouse, rat sheep,goat, hamster, rabbit, etc. The origin of the protein immunogen may bemouse, human, rat, monkey, etc or a microorganism, including a bacteriaor a virus. The host animal will generally be a different species thanthe immunogen, e.g. human protein used to immunize mice, etc.

[0101] The immunogen may comprise the complete protein, or fragments andderivatives thereof. Preferred immunogens comprise all or a part of oneof the subject proteins, where these residues contain thepost-translation modifications, such as glycosylation, found on thenative target protein. Immunogens comprising the extracellular domainare produced in a variety of ways known in the art, e.g. expression ofcloned genes using conventional recombinant methods, isolation fromtumor cell culture supernatants, etc.

[0102] For preparation of polyclonal antibodies, the first step isimmunization of the host animal with the target protein, where thetarget protein will preferably be in substantially pure form, comprisingless than about 1% contaminant. The immunogen may comprise the completetarget protein, fragments or derivatives thereof. To increase the immuneresponse of the host animal, the target protein may be combined with anadjuvant, where suitable adjuvants include alum, dextran, sulfate, largepolymeric anions, oil & water emulsions, e.g. Freund's adjuvant,Freund's complete adjuvant, and the like. The target protein may also beconjugated to synthetic carrier proteins or synthetic antigens. Avariety of hosts may be immunized to produce the polyclonal antibodies.Such hosts include rabbits, guinea pigs, rodents, e.g. mice, rats,sheep, goats, and the like. The target protein is administered to thehost, usually intradermally, with an initial dosage followed by one ormore, usually at least two, additional booster dosages. Followingimmunization, the blood from the host will be collected, followed byseparation of the serum from the blood cells. The Ig present in theresultant antiserum may be further fractionated using known methods,such as ammonium salt fractionation, DEAE chromatography, and the like.

[0103] Monoclonal antibodies are produced by conventional techniques.Generally, the spleen and/or lymph nodes of an immunized host animalprovide a source of plasma cells. The plasma cells are immortalized byfusion with myeloma cells to produce hybridoma cells. Culturesupernatant from individual hybridomas is screened using standardtechniques to identify those producing antibodies with the desiredspecificity. Suitable animals for production of monoclonal antibodies tothe human protein include mouse, rat, hamster, etc. To raise antibodiesagainst a mouse protein, the animal will generally be a hamster, guineapig, rabbit, etc. The antibody may be purified from the hybridoma cellsupernatants or ascites fluid by conventional techniques, e.g. affinitychromatography using protein according to the subject invention bound toan insoluble support, protein A sepharose, etc.

[0104] The antibody may be produced as a single chain, instead of thenormal multimeric structure. Single chain antibodies are described inJost et al. (1994) J.B.C. 269:26267-73, and others. DNA sequencesencoding the variable region of the heavy chain and the variable regionof the light chain are ligated to a spacer encoding at least about 4amino acids of small neutral amino acids, including glycine and/orserine. The protein encoded by this fusion allows assembly of afunctional variable region that retains the specificity and affinity ofthe original antibody.

[0105] Also provided are “artificial” antibodies, e.g., antibodies andantibody fragments produced and selected in vitro. In some embodiments,such antibodies are displayed on the surface of a bacteriophage or otherviral particle. In many embodiments, such artificial antibodies arepresent as fusion proteins with a viral or bacteriophage structuralprotein, including, but not limited to, M13 gene III protein. Methods ofproducing such artificial antibodies are well known in the art. See,e.g., U.S. Pat. Nos. 5,516,637; 5,223,409; 5,658,727; 5,667,988;5,498,538; 5,403,484; 5,571,698; and 5,625,033.

[0106] For in vivo use, particularly for injection into humans, it isdesirable to decrease the antigenicity of the antibody. An immuneresponse of a recipient against the blocking agent will potentiallydecrease the period of time that the therapy is effective. Methods ofhumanizing antibodies are known in the art. The humanized antibody maybe the product of an animal having transgenic human immunoglobulinconstant region genes (see for example International Patent ApplicationsWO 90/10077 and WO 90/04036). Alternatively, the antibody of interestmay be engineered by recombinant DNA techniques to substitute the CH1,CH2, CH3, hinge domains, and/or the framework domain with thecorresponding human sequence (see WO 92/02190).

[0107] The use of Ig cDNA for construction of chimeric immunoglobulingenes is known in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987)J. Immunol. 139:3521). mRNA is isolated from a hybridoma or other cellproducing the antibody and used to produce cDNA. The cDNA of interestmay be amplified by the polymerase chain reaction using specific primers(U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library ismade and screened to isolate the sequence of interest. The DNA sequenceencoding the variable region of the antibody is then fused to humanconstant region sequences. The sequences of human constant regions genesmay be found in Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, N.I.H. publication no. 91-3242. Human C regiongenes are readily available from known clones. The choice of isotypewill be guided by the desired effector functions, such as complementfixation, or activity in antibody-dependent cellular cytotoxicity.Preferred isotypes are IgG1, IgG3 and IgG4. Either of the human lightchain constant regions, kappa or lambda, may be used. The chimeric,humanized antibody is then expressed by conventional methods.

[0108] In yet other embodiments, the antibodies may be fully humanantibodies. For example, xenogeneic antibodies which are identical tohuman antibodies may be employed. By xenogenic human antibodies is meantantibodies that are the same has human antibodies, i.e. they are fullyhuman antibodies, with exception that they are produced using anon-human host which has been genetically engineered to express humanantibodies. See e.g. WO 98/50433; WO 98,24893 and WO 99/53049, thedisclosures of which are herein incorporated by reference.

[0109] Antibody fragments, such as Fv, F(ab′)₂ and Fab may be preparedby cleavage of the intact protein, e.g. by protease or chemicalcleavage. Alternatively, a truncated gene is designed. For example, achimeric gene encoding a portion of the F(ab′)₂ fragment would includeDNA sequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

[0110] Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

[0111] Expression vectors include plasmids, retroviruses, YACs, EBVderived episomes, and the like. A convenient vector is one that encodesa functionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g. SV-40 earlypromoter, (Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcomavirus LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murineleukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native Igpromoters, etc

[0112] An example of a disease that can be prevented or treatedutilizing the present invention is CMV infection. The results presentedin the examples demonstrate for the first time a role for DC-SIGN in CMVbinding to human dendritic cells.

[0113] The experiment described herein, including the results describedin the examples, provide new insights into the mechanisms of interactionof CMV with DC and the transmission of CMV infection to other celltargets. The data show that DC-SIGN accounts for most of the binding ofCMV to DC and mediates the attachment of CMV virions when expressed inDC-SIGN negative cells. Interaction of CMV with DC-SIGN occurs throughspecific binding with at least one CMV envelope glycoprotein, CMV gB.DC-SIGN is a type II membrane protein in which the extracellular domainencompass the CRD and a stalk that mediates tetramerization (Mitchell etal., 2001). Like DC-SIGN, CMV gB is also present in multimeric complexesin CMV envelope (Scheffczik et al., 2001). CMV gB-DC-SIGN interactionsanalysed by SPR were conducted using recombinant soluble forms ofDC-SIGN CRD, which are monomers. The affinity of CMV gB for DC-SIGNmeasured by SPR was 0.3 PM and was comparable to that estimated forHIV-1 gp120 (Mitchell et al., 2001). This relatively low affinity islikely due to the inability of CRD to multimerize. The estimatedaffinity (Kd) of HIV-1 gp120 for the natural DC-SIGN molecule is 1.4 nM(versus 5 nM for CD4) (Curtis et al., 1992). These findings suggestthat, like HIV-1 gp120, CMV gB would display high affinity foroligomerized DC-SIGN.

[0114] DC-SIGN-bound CMV retains infectious capacity since, upon bindingonto DC-SIGN⁺ THP-1 cells or MD-DC, CMV is transmitted to permissivecells where the virus replicates actively. DC are receptive to CMVinfection by primary, non-adapted CMV isolates and refractory toinfection by adapted, CMV laboratory strains. The capacity of DC totransmit CMV to permissive cell targets can be dissociated from theability of CMV to infect and replicate in DC. This result was confirmedusing DC-SIGN⁺ HeLa cells which were capable of transmitting CMV topermissive target cells while CMV IE antigen expression was neverdetected in these refractory cells. This DC-SIGN function is reminiscentof the aptitude to transmit infection to CD4⁺ T lymphocytes shown eitherby HIV-1-pulsed-DC or -DC-SIGN-transduced cells.

[0115] Binding to, and transfer of HIV-1 from, DC-SIGN⁺ cells appear tobe separable steps (Pohlmann et al., 2001a). Recently, it has been shownthat efficient transmission of HIV to CD4⁺ T lymphocytes fromDC-SIGN-expressing THP-1 cells requires internalization signals encodedin the cytoplasmic domain of the lectin (Kwon et al., 2002). Therequirement of DC-SIGN cytoplasmic signals for efficient trans-infection(named trans-enhancement) becomes particularly evident when low amountof virus are used as inoculum (Geijtenbeek et al., 2000b). Similarly toHIV, sub-optimal inoculums of CMV become highly infectious whentransferred from DC-SIGN⁺ THP-1 cells. Moreover, DC-SIGN Δ35 and Δ20failed to support CMV transmission to highly susceptible cells andincubation of CMV with wild type- or truncated-DC-SIGN-expressing cellsat 4° C. prevented CMV transmission to permissive cells. These findingssuggest that endocytosis of the receptor is required for efficienttransmission of CMV to permissive cells. However, the experimentsreported here do not permit to rule out the involvement of putativetransduction of intracellular signals in this phenomenon since deletionof DC-SIGN cytoplasmic domains or inhibition of cell signal activationat 4° C. may preclude DC-SIGN-dependent cell activation. Overall, thesefindings suggest that in the natural CMV infection, DC-SIGN promotestake up of CMV and permits enhancement of CMV transmission byinterstitial DC to other cells. The hypothesis of in vivo CMV transportby DC raised the question of the stability of DC-SIGN-bound CMVparticles. As previously described for HIV, the experiments describedherein provide evidence that DC-SIGN⁺ THP-1 cells can transmit CMV toother cell targets after five days in culture whereas cell-free virusloss infectivity upon incubation at 37° C. for 24 to 48 hours. Theability of DC to transmit infection long time after exposure supportsthe hypothesis that DC transport small amounts of CMV from entry sitesto target organs where they could transmit infectious CMV particles bycell-to-cell contact.

[0116] A striking feature of DC-SIGN-CMV interactions is the capacity ofthe lectin to facilitate the infection of low-susceptible cells to CMVinfection. Thus, THP-1 cells that do not normally support CMVreplication become productively infected as they express DC-SIGN. CMVattachment to host cells is supposed to occur namely through lowaffinity interactions with heparan sulfate proteoglycans (Compton etal., 1993; Kari and Gehrz, 1992). However, beyond this primary site ofbinding, the existence of an alternative cellular co-factor, requiredfor a strong attachment of CMV on cell membranes as well as for itsentry into cells, is postulated (Boyle and Compton, 1998). Annexin II,which binds to CMV gB (Pietropaolo and Compton, 1997) and a 92.5 kDaprotein which binds to CMV gH (Baldwin et al., 2000), have been proposedto play this role. It is unlikely that DC-SIGN is the elusive CMVreceptor that ultimately determines entry of the enveloped virions andreplication in CMV-infection susceptible cells. Indeed, CMV entry andinfection occurs in a number of cell types (i.e., MRC-5 fibroblasts andU373 MG astrocytoma cells) where DC-SIGN is not expressed. The putativeCMV receptor in these cells might be different from a lectin, althoughthe existence of yet unidentified DC-SIGN-like molecules accounting forbinding and entry of CMV cannot be formally ruled out.

[0117] The capacity of DC-SIGN to promote in cis CMV replication inotherwise low-susceptible cells may result from any of three notmutually exclusive hypotheses. DC-SIGN has the capacity to capture andinternalize HIV-1 in DC (Kwon et al., 2002). By analogy, DC-SIGN mightpromote internalization and trafficking of CMV to an intracellularcompartment from where it could initiate the infectious cycle.Alternatively, attachment of CMV to DC-SIGN, or DC-SIGNR, mightfacilitate the interaction with the authentic cellular receptor, whichultimately would account for CMV entry. Such a function would bereminiscent of the facilitating effect shown by DC-SIGN on HIV infectionof T lymphocytes displaying low levels of CCR5 (Lee et al., 2001).Finally, differentiation of THP-1 cells with TPA was shown to inducepermissiveness to CMV replication (Weinshenker et al., 1988). Similarly,signal transduction through DC-SIGN could lead to cellulardifferentiation and subsequent CMV replication.

[0118] Regarding CMV infection, the in cis capacity of DC-SIGN tofacilitate viral entry is likely of biological relevance since theblockade by specific anti-DC-SIGN antibodies drastically reducesinfectiveness of DC by primary, CMV isolates. The capacity of DC tosupport CMV infection may be related to the amount of DC-SIGN expressedat their surface. Thus, immature DC which express high levels of DC-SIGNcan be infected by CMV (Raftery et al., 2001; Riegler et al., 2000)while matured DC that display low DC-SIGN expression show reducedsusceptibility to CMV. Expression of DC-SIGN on immature DC ofintestinal and genital mucosae (Geijtenbeek et al., 2000b; Jameson etal., 2002) may confer to this co-factor a crucial role for the infectionof these primary target cells at the anatomical sites where initial CMVtransmission or propagation most probably take place. A recent studydescribed a monocyte-derived macrophage circulating subset, expressingDC markers in vivo (Soderberg-Naucler et al., 1997). This subset wasshown to harbor latent CMV which reactivates upon allogeneicstimulation. It appears necessary to investigate the expression ofDC-SIGN by these cells which could represent a biological link betweenthis newly identified dendritic-like subset and the results describedherein. As recently reported, CMV infected DC display decreased antigenpresentation and differentiation capacities (Andrews et al., 2001;Raftery et al., 2001). Hence, by promoting DC mediated trans-infectionof target cells as well as cis-infection of DC, DC-SIGN could beinvolved, apart from virus propagation, in CMV-mediated altered immuneresponse.

[0119] The data reported herein show that DC-SIGNR is also able to bindCMV gB and to promote cis-infection of apparently low susceptible cells.This DC-SIGN homologue is mainly expressed on EC (Bashirova et al.,2001; Pohlmann et al., 2001b) which are known to be preferential targetsof CMV in vivo and replicate primary, non-adapted CMV strains in vitro(Kahl et al., 2000). The expression of DC-SIGNR on placental EC andmacrophages (Soilleux et al., 2001) could be involved in thematerno-fetal transmission of CMV during congenital infections.Similarly, DC-SIGNR expressed in liver EC may be implicated inCMV-induced hepatitis, one of the most frequent clinical forms of thisinfection.

[0120] Murine CMV shares many essential characteristics with its humancounterpart and has been a widely studied model for CMV infection. Ithas been shown that infection of DC by murine CMV prevents delivery ofthe signals required for T cell activation. The impairment of DCfunctions by murine CMV is supposed to be detrimental for the hostimmune responses (Andrews et al., 2001). The cloning of severalhomologues of DC-SIGN in mice (Park et al., 2001), should provide thismodel with an invaluable tool for studying the implication ofDC-SIGN-like molecules in the dynamic of CMV dissemination, the role ofthe different subsets of DC in the course of CMV propagation andeventually the causes of CMV-induced immunosuppression.

[0121] In accordance with these results, the invention provides a methodof preventing or treating a disease of a mammal, where at least onesymptom of the disease is mediated at least in part by the binding of aneffector molecule to a DC-SIGN receptor of the mammal to be treated, andwhere the method comprises administering to the mammal an amount of aDC-SIGN blocker sufficient to substantially inhibit the binding of theeffector molecule to the DC-SIGN receptor to thereby prevent or treatthe disease.

[0122] In some embodiments the DC-SIGN blocker is a blocking derivativeof the effector molecule. In other embodiments the DC-SIGN blocker is anantibody.

[0123] Among embodiments of the invention where the DC-SIGN blocker isan antibody are included embodiments where the antibody specificallybinds DC-SIGN and embodiments where the antibody specifically binds theeffector molecule.

[0124] In some embodiments the DC-SIGN blocker is a mannosylatedmolecule that binds to a DC-SIGN receptor. The mannosylated molecule maybe mannan.

[0125] The invention also provides a method of preventing or treating aviral infection of a mammal, where the viral infection is mediated atleast in part by the binding of a viral effector molecule to a DC-SIGNreceptor of the mammal to be treated, where the method comprisesadministering to the mammal an amount of a DC-SIGN blocker sufficient tosubstantially inhibit the binding of the viral effector molecule to theDC-SIGN receptor to thereby prevent or treat the viral infection.

[0126] In some embodiments of the method the DC-SIGN blocker comprises abinding moiety of the viral effector molecule. In other embodiments theDC-SIGN blocker comprises a binding moiety of a viral envelopeglycoprotein. In other embodiments the DC-SIGN blocker is an antibody.The antibody may specifically bind DC-SIGN or specifically bind theviral effector molecule. In additional embodiments the DC-SIGN blockeris a mannosylated molecule that binds to a DC-SIGN receptor. Themannosylated molecule may be mannan.

[0127] Among embodiments of the invention in which the DC-SIGN blockeris an antibody are included embodiments in which the antibody is amonoclonal antibody; the mammal is a human and the antibody is amonoclonal antibody that is humanized; the antibody specifically bindsDC-SIGN; the monoclonal antibody is Mab 1B10.2.6; the antibodyspecifically binds the viral effector molecule; and the antibodyspecifically binds the binding moiety of the viral effector molecule.

[0128] In further embodiments of the method the viral effector moleculeis a molecular constituent of the viral envelope. In certain embodimentsthe molecular constituent of the viral envelope is an envelopeglycoprotein.

[0129] In additional embodiments of the method the DC-SIGN blockercomprises a binding moiety of the viral effector molecule. In someembodiments of the invention in which the viral effector molecule is amolecular constituent of the viral envelope the DC-SIGN blocker that isused comprises a binding moiety of the envelope glycoprotein.

[0130] In a preferred embodiment, the viral infection is a CMV infectionand the viral effector molecule is a CMV effector molecule. In a furtherpreferred aspect the mammal is a human. In some embodiments the CMVeffector molecule is a molecular constituent of the CMV envelope. Infurther embodiments the molecular constituent of the CMV envelope is aCMV envelope glycoprotein. In yet further embodiments the CMV envelopeglycoprotein is CMV envelope glycoprotein B.

[0131] Included among embodiments of the invention in which the viralinfection is a CMV infection and the viral effector molecule is a CMVeffector molecule are embodiments where the DC-SIGN blocker comprises abinding moiety of the CMV effector molecule; the DC-SIGN blockercomprises a binding moiety of the CMV envelope glycoprotein B; theDC-SIGN blocker is a recombinantly produced protein; and the DC-SIGNblocker is an antibody. Among embodiments where the DC-SIGN blocker isan antibody are embodiments where the antibody is a monoclonal antibody;the mammal is a human and the monoclonal antibody is humanized; theantibody specifically binds DC-SIGN; the monoclonal antibody is Mab1B10.2.6; and the antibody specifically binds the CMV effector molecule.Among embodiments where the antibody specifically binds the CMV effectormolecule are embodiments where the CMV effector molecule is CMV envelopeglycoprotein B.

[0132] In one preferred embodiment of the invention the effectormolecule and the DC-SIGN blocker are the same. In a second preferredembodiment the effector molecule and the DC-SIGN blocker are different.

[0133] It is interesting that CMV, Ebola, and HIV (as well as SIV) canbind to DC-SIGN. HIV binding to dendritic cells is mediated by thebinding of the gp120 glycoprotein of HIV with DC-SIGN. Thus, gp120 is aviral effector molecule. The invention thus provides a method for theprevention and treatment of an Ebola invention and an HIV infection.Specifically, it is an object of the invention to provide a method ofpreventing or treating an Ebola, HIV or SIV infection of a human or asimian. The method comprises administering to the human or simian anamount of a DC-SIGN blocker that is sufficient to inhibit theinteraction of Ebola, HIV or SIV with DC-SIGN receptor present ondendritic cells of the human or simian to thereby prevent or treat theEbola, HIV or SIV infection.

[0134] DC-SIGN is also believed to have a critical role in mediating theknown loose adhesion that takes place between dendritic cells and Tcells in the apparent absence of foreign antigen. This adhesion isthought to be necessary to provide an opportunity for the TCR to scanthe dendritic cell surface and identify the very small amounts of TCRligand which are present, and in turn to become activated by thisligand. For this reason, the interaction between DC-SIGN on dendriticcells, and ICAM-3 on T cells, is likely to be critically important forthe process of T cell activation and stimulation. This model suggeststhat the DC-SIGN-ICAM-3 interaction may have a role in mediating and/orpotentiating other stimulatory effects of dendritic cells on T cells.

[0135] For this reason DC-SIGN blockers may be potent anti-inflammatoryagents, by blocking the interaction of the ICAM-3 effector molecule withDC-SIGN. Accordingly, the invention also provides a method of preventingor treating inflammation in a mammal caused by interaction of ICAM-3present on T cells of the mammal with DC-SIGN receptor present ondendritic cells of the mammal. The method comprises administering to themammal an amount of a DC-SIGN blocker that is sufficient to inhibit theinteraction of ICAM-3 present on T cells of the mammal with DC-SIGNreceptor present on dendritic cells of the mammal to thereby prevent ortreat inflammation.

[0136] The invention also provides pharmaceutical compositionscomprising a DC-SIGN blocker. Such compositions may be suitable forpharmaceutical use and administration to patients. The compositionstypically contain a purified DC-SIGN blocker at a therapeuticallyachievable concentration and a pharmaceutically acceptable excipient. Asused herein, the phrase “pharmaceutically acceptable excipient” includesany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, that are compatible with pharmaceutical administration. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. The compositions can also contain other activecompounds providing supplemental, additional, or enhanced therapeuticfunctions. The pharmaceutical compositions can also be included in acontainer, pack, or dispenser together with instructions foradministration.

[0137] A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Methods toaccomplish the administration are known to those of ordinary skill inthe art. The administration may, for example, be intravenous,intramuscular, subcutaneous, or via inhalation.

[0138] Solutions or suspensions used for subcutaneous applicationtypically include one or more of the following components: a sterilediluent, such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerin, propylene glycol or other syntheticsolvents; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite;chelating agents, such as ethylenediaminetetra acetic acid; buffers,such as acetates, citrates or phosphates; and agents for the adjustmentof tonicity, such as sodium chloride or dextrose. The pH can be adjustedwith acids or bases, such as hydrochloric acid or sodium hydroxide. Suchpreparations can be enclosed in ampoules, disposable syringes ormultiple dose vials made of glass or plastic.

[0139] Pharmaceutical compositions suitable for injection includesterile aqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersion. For intravenous administration, suitable carriers includephysiological saline, bacteriostatic water, Cremophor ELTM (BASF,Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, thecomposition must be sterile and should be fluid to the extent that easysyringability exists. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The carrier can bea solvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), and suitable mixtures thereof. The properfluidity can be maintained, for example, by the use of a coating such aslecithin, by the maintenance of the required particle size in the caseof dispersion and by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, one may include isotonicagents, for example, sugars, polyalcohols such as manitol, sorbitol,sodium chloride in the composition. Prolonged absorption of theinjectable compositions can be brought about by including in thecomposition an agent which delays absorption, for example, aluminummonostearate and gelatin.

[0140] Systemic administration can also be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants are generally known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and fusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

[0141] For administration by inhalation, the DC-SIGN blocker containingcompositions are delivered in the form of an aerosol spray frompressured container or dispenser, which contains a suitable propellant,e.g., a gas such as carbon dioxide, or a nebulizer.

[0142] In one embodiment, a purified DC-SIGN blocker is prepared withcarriers that will protect it against rapid elimination from the body,such as a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensionscontaining LAM can also be used as pharmaceutically acceptable carriers.These can be prepared according to methods known to those skilled in theart, for example, as described in U.S. Pat. No. 4,522,811.

[0143] Therapeutically useful agents, such as growth factors (e.g.,BMPs, TGF-β, FGF, IGF), cytokines (e.g., interleukins and CDFs),antibiotics, and any other therapeutic agent beneficial for thecondition being treated can optionally be included in or administeredsimultaneously or sequentially with the DC-SIGN blocker.

[0144] It is especially advantageous to formulate compositions in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the subject to be treated; each unit containing apredetermined quantity of active compound calculated to produce thedesired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the uniquecharacteristics of the active compound and the particular therapeuticeffect to be achieved, and the limitations inherent in the art ofcompounding such an active compound for the treatment of individuals.

[0145] Toxicity and therapeutic efficacy of compositions comprising aDC-SIGN blocker can be determined by standard pharmaceutical proceduresin cell cultures or experimental animals, e.g., for determining the LD50(the dose lethal to 50% of the population) and the ED50 (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD50/ED50. DC-SIGN blockers which exhibitlarge therapeutic indices are preferred.

[0146] The data obtained from the cell culture assays and animal studiescan be used in formulating a range of dosage for use in humans. Thedosage of such compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any DC-SIGNblocker used in the present invention, the therapeutically effectivedose can be estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test DC-SIGN blocker which achieves a half-maximal inhibition ofsymptoms) as determined in cell culture. Levels in plasma can bemeasured, for example, by high performance liquid chromatography. Theeffects of any particular dosage can be monitored by a suitablebioassay.

[0147] A targeting complex of the present invention comprises at leastone DC-SIGN blocker molecule covalently attached to at least one subjectmolecule. In some embodiments, a single DC-SIGN blocker molecule iscovalently linked to a single subject molecule. In other embodiments,more than one DC-SIGN blocker molecule can be covalently linked to asingle subject molecule. The multiple DC-SIGN blocker molecules can eachbe independently covalently linked to the subject molecule;alternatively, one or more of the more than one DC-SIGN blockermolecules can be covalently linked only to one or more other DC-SIGNblocker molecules, at least one of which is itself covalently linked tothe subject molecule.

[0148] In other embodiments, multiple subject molecules are covalentlylinked to a single DC-SIGN blocker molecule. The multiple subjectmolecules can each be independently covalently linked to the DC-SIGNblocker molecule; alternatively, one or more of the more than onesubject molecules can be covalently linked only to one or more othersubject molecules, at least one of which is itself covalently linked tothe DC-SIGN blocker molecule.

[0149] Additional embodiments of the invention utilize compositions ofmore than one of the various types of DC-SIGN blockers describedimmediately above. There is no limit to the diversity of suchcompositions which can be used. One of skill in the art will appreciatethat the composition to be used for a particular application will bedictated by many factors and that a suitable composition can thus beappropriately chosen for each application of the invention.

[0150] Techniques for making the DC-SIGN blockers of the invention arewell known and widely practiced by those of skill in the biochemistryart, and thus need not be detailed here. However, one of skill in theart will recognize that any suitable technique which results in theformation of a covalent bond between a subject molecule and a DC-SIGNblocker molecule can be used.

[0151] Subject molecules can be any molecule of interest. Nonlimitingexamples include: small organic molecules, proteins, nucleic acids,carbohydrates, and lipids. One of ordinary skill in the art willappreciate that any known derivatives and composites of one or more ofthese classes of molecules can also be used.

[0152] In the case in which the subject molecule is a protein, nucleicacid, carbohydrate, or lipid, the subject molecule can be obtained froma natural source, i.e., purified from an organism, which comprises themolecule. Alternatively, the subject molecule can be obtained from arecombinant source, i.e., from a recombinant organism, which has beenengineered to produce a subject molecule of choice. In some cases, therecombinant organism that is used to produce the subject molecule is onethat comprises the subject molecule, as the organism occurs in nature,in nonrecombinant form. In other cases, the subject molecule is one thatdoes not naturally occur in the recombinant organism.

[0153] The subject molecules of the invention also include derivativesof small organic molecules, proteins, nucleic acids, carbohydrates, andlipids. As used here, a derivative is a form of small organic molecule,protein, nucleic acid, carbohydrate, or lipid that is modified from itsnatural state by adding, subtracting, or altering one or more chemicallyreactive sites present on the small organic molecule, protein, nucleicacid, carbohydrate, or lipid. Techniques for making derivatives of smallorganic molecules, proteins, nucleic acids, carbohydrates, and lipidsare well known and widely practiced by those of skill in thebiochemistry art, and thus need not be detailed here.

[0154] In a preferred embodiment the subject molecule is an antibody.

[0155] The subject molecule can also be a molecule that is antigenic. Amolecule is antigenic when it is capable of specifically interactingwith an antigen recognition molecule of the immune system, such as animmunoglobulin (antibody) or T cell antigen receptor. An antigenicpolypeptide contains at least about 5, and preferably at least about 10,amino acids. An antigenic portion of a molecule can be that portion thatis immunodominant for antibody or T cell receptor recognition, or it canbe a portion used to generate an antibody to the molecule by conjugatingthe antigenic portion to a carrier molecule for immunization. A moleculethat is antigenic need not be itself immunogenic, i.e., capable ofeliciting an immune response without a carrier.

[0156] The targeting complex of the invention can be exposed to adendritic cell either in vivo or in vitro. In vivo exposure is achievedby administering the targeting complex in a pharmaceutical compositionas described herein or in any suitable equivalent formulation known inthe art. In that case, the targeting complex will bind to DC-SIGN on thesurface of dendritic cells in vivo. In vitro exposure occurs whendendritic cells grown in vitro are exposed to the targeting complex.

[0157] The following examples aid in describing certain aspects of theinvention. One of ordinary skill in the art will recognize the numerousmodifications and variations that may be performed without altering thespirit or scope of the present invention. Such modifications andvariations are believed to be encompassed within the scope of theinvention. The examples do not in any way limit the invention.

EXAMPLES Example 1 Herpesviruses

[0158] AD169, Towne (CMV laboratory strains) and TB40/E (CMV clinicalisolate) were provided by Dr. S. Michelson (Institut Pasteur, Paris,France) and Dr. C. Sinzger (Tubingen, Germany), respectively. ADGFP is agenetically modified AD169 strain encoding an Enhanced Green FluorescentProtein driven by the CMV immediate-early gene promoter (Borst et al.,2001). VZV and HSV-1 clinical isolates were obtained from Dr. IsabelleGarrigue (Laboratory of Virology, CHU Pellegrin, Bordeaux, France).

Example 2 Reagents, Antibodies and Viral Glycoproteins

[0159] Mannan and EGTA were purchased from Sigma-Aldrich Corporation(Saint Louis, Mo.). Soluble viral envelope glycoproteins were producedand purified from mammalian or insect cells. HIV-1 gp120 (MN isolate)was obtained from the NIBSC repository (Medical Research Council, UnitedKingdom). VZV gB and VZV gE (Jacquet et al., 1995) were gifts from Dr.A. Jacquet, (Department of Applied Genetics, Gosselies, Belgium). HSV-1gB and HSV-1 gD (Sisk et al., 1994) were provided by Dr. G. H. Cohen(University of Pennsylvania, Philadelphia, Pa.). Expression andpurification of CMV gB (gift of Dr Claude Meric, Aventis Pasteur, MarcyL'Etoile, France) were previously described (Norais et al., 1996; Passet al., 1999). Anti-CMV gB (clone 1-M-12, IgG1) and anti-DC-SIGNR (clone120604, IgG2a) mAbs were purchased from Biodesign International (Saco,Me.) and R&D Systems (Minneapolis, Minn.), respectively. Anti-LIF 7D2(Taupin et al., 1993) and anti-SDF-1 K15C monoclonal antibodies (mAb)(Amara et al., 1999) were used as isotypic controls.

Example 3 Cells

[0160] MRC-5 (Bio Merieux S.A., Marcy l'Etoile, France) and U373 MG(ECCC, Salisbury, United Kingdom) are CMV-, HSV-1 - and VZV-permissivecell lines, from fibroblastic and astrocytic origine, respectively.Parental and DC-SIGN⁺ THP-1 cells (wild type and Δ35 and Δ20 mutantslacking the first 35 and 20 amino acids of the cytoplasmic domain,respectively) (Kwon et al., 2002) were a gift from Dr. D. R. Littmann(Skirball Institute of Biomolecular Medicine, New York, N.Y., USA).DC-SIGN⁺ HeLa cells were generated by infecting HeLa cells with anHIV-derived vector (TRIP-AU3 vector, a gift from Dr. P. Charneau,Institut Pasteur, Paris) encoding a human DC-SIGN cDNA. MD-DC weregenerated from peripheral blood monocytes treated with 20 ng/mL IL-4(Schering-Plough, Kenilworth, N.J.) and 100 ng/mL GM-CSF (Leucomax,Novartis-Pharma, Rueil Malmaison, France) (Romani et al., 1994). At day5, virtually the totality of cells displayed the phenotype CD1a⁺,HLA-DR⁺,CD80^(low), CD86^(low), CD83⁻, CD14⁻ characteristic of immatureMD-DC.

Example 4 DC-SIGN cDNA and anti-DC-SIGN Antibodies

[0161] DC-SIGN cDNA was isolated from human immature MD-DC by RT-PCR.For expression in mammalian cells, human DC-SIGN was subcloned at theEcoR1/Xba 1 sites of the pcDNA3 myc-His (version A) plasmid (Invitrogen,Carsbad, Calif.). The DC-SIGNR cDNA was a gift from Dr R. W. Doms,(University of Pennsylvania, Philadelphia, Pa.). Anti-DC-SIGN clone1B10.2.6 (IgG2a) was obtained by immunizing BALB/c mice with HEK 293Tcells transfected with DC-SIGN cDNA, screened by indirectimmunofluorescent staining and FACS analysis on DC-SIGN⁺ HeLa cells andused as purified immunoglobulins.

Example 5 Infection Assays

[0162] For trans-infection experiments, cells were incubated with viralsuspensions (CMV, VZV or HSV-1, MOI=1) for 2 hr, at 37° C. Thereafter,unbound viral particles were removed by extensive washes and cells wereco-cultured with sub-confluent MRC-5 or U373 MG cell monolayers. After24 to 72 hr, infected MRC-5 or U373-MG cells were fixed, permeabilizedand stained with specific mAbs directed against IEA- or EA-CMV (mAbs E13and 2A2, respectively), VZV (mAb 2013) or HSV (mAb CHA-437) (ArgenBiosoft, Varilhes, France). When indicated MD-DC or THP-1 (parental orDC-SIGN⁺) cells were incubated with EGTA (5 mM), mannan or anti-DC-SIGN(1B10.2.6 mAb) for 30 minutes at 4° C. prior challenge with infectiouspreparations. Infection by ADGFP strain was assessed by countingGFP-expressing cells at day 3. For long term infectivity experimentsDC-SIGN⁺ or parental THP-1 cells were incubated with ADGFP (MOI=1) for 4hr at 37° C. After extensive washes, infected cells were incubated at37° C. and an aliquot of these cells was added to a sub-confluent MRC-5cell culture every 2 days during the assay.

[0163] To assess the effect in cis of DC-SIGN during infection, cellswere incubated with low titers of CMV (MOI=0.1) for 2 hr at 37° C. Noninternalized viral particles were removed by washes in low pH citratebuffer (pH=3). The number of infected cells was determined byimmunocytochemistry 72 hours after infection. Supernatants from infectedcells kept in culture for 14 days were harvested to quantify de novogenerated virions by plaque-assay titration.

Example 6 HIV-1 gp120 Binding Competition and CMVgB Direct BindingAssays

[0164] DC-SIGN⁺ THP-1 cells were washed two times, resuspended inice-cold binding buffer (1 mM CaCl₂, 2 mM MgCl₂ and 0.1% Bovine SerumAlbumin in PBS) at 10⁶ cells/mL and pre-treated or not for 15 minuteswith competitors (20 μg/ml). Thereafter, recombinant biotinylatedCXCR4-tropic (MN isolate) HIV-1 gp120 (2μg/ml; Immunodiagnostics Inc.,Woburg, Mass.) was added for 30 minutes at 4° C. After extensivewashing, cell-bound biotinylated HIV-1 gp120 was revealed by flowcytometry using FITC-conjugated Streptavidin (Immunotech SA, Marseille,France). For CMV gB binding experiments, recombinant soluble CMV gB andBovine Serum Albumine (BSA; Amersham Pharmacia Biotech, Uppsala, Sweden)were biotinylated with sulfo-NHS biotin, according to manufacturerinstructions (Pierce, Rockford, Ill.).

Example 7 Analysis of DC-SIGN Interactions with Viral EnvelopeGlycoproteins by SPR

[0165] The cDNA coding for the DC-SIGN CRD (amino acids 254-404) wasobtained by PCR and cloned into pET15b (Novagen). The protein wasexpressed in Escherichia coli C41 (DE3) as inclusion bodies. Refoldingof the protein has been done by dilution and dialysis as described(Mitchell et al., 2001). Purification of refolded DC-SIGN CRD has beenachieved in two steps: first on a Ni-NTA (QIAGEN) column equilibrated in25 mM Tris Cl pH 7.8, 150 mM NaCl, 4 mMCaCl₂ (Loading Buffer) and elutedwith a linear gradient of imidazole and second on a Mannose-agarosecolumn equilibrated in Loading Buffer, and eluted in buffer where CaCl₂was replaced by EDTA (10 mM). Pooled fractions are then concentrated anddialyzed against Loading Buffer.

[0166] Four flow cells of a Biacore B1 sensor chip were activated asdescribed (Amara et al., 1999). The first flow cell was then blockedwith 50 μl of 1 M.ethanolamine pH 8.5 and served as a control surface.The three other ones were treated with soluble gp120, gB CMV or gB HSV(concentration range 1-10 μg/ml in 10 mM acetate buffer pH 5).Typically, this procedure permitted the coupling of approximately250-350 resonance units (RU) of proteins. For binding assays, DC-SIGNCRD was diluted in Loading Buffer and was allowed to react with thesensor chip (at 30 μl/min). In a typical analysis, DC-SIGN CRD (0.13 to1 μM, see figure legend) was injected over the four flow cells for 8min, after which the complexes were rinsed with buffer to analyze thedissociation phase. The surface was then regenerated with a 6 min pulseof running buffer containing 50 mM EDTA instead of CaCl₂. Sets ofsensorgrams were analyzed using the BIAevaluation 3 software.

Example 8 Expression of DC-SIGN at the Cell Membrane Enables Binding ofCMV

[0167] The capacity of CMV to bind DC-SIGN was investigated. Parentaland DC-SIGN⁺ THP-1 cells, or immature monocyte-derived DC (MD-DC) wereincubated on ice with increasing concentrations of CMV and the presenceof cell-bound virions was quantified by flow cytometry using a mAbdirected against the CMV gB. While parental THP-1 cells failed to binddetectable amounts of CMV, both DC-SIGN-expressing THP-1 and MD-DCabsorbed CMV virions in a dose dependent manner (FIG. 1B). Prevention ofCMV gB antibody-labeling by acidic washes proved the existence ofcell-bound virions (FIG. 1B). Abrogation of virion attachment observedfollowing pre-incubation of cells with mannan, a complex sugar thatbinds to the Carbohydrate Recognition Domain (CRD) of lectins, suggeststhat the CMV-DC-SIGN interaction is accounted by the glycosylatedresidues of CMV envelope glycoproteins.

Example 9 Transmission of CMV Infection to Permissive Cells is Mediatedby DC-SIGN

[0168] MD-DC, THP-1 or HeLa expressing DC-SIGN were incubated with amutant CMV strain encoding a GFP (ADGFP) (Borst et al., 2001). HeLacells were selected for their refractoriness to CMV infection (Einhornet al., 1982; Tsutsui et al., 1987) which persists despite transductionwith DC-SIGN (our unpublished observations). MD-DC (FIG. 2A), DC-SIGN⁺THP-1 (FIG. 2B) and DC-SIGN⁺ HeLa cells (FIG. 2C), in contrast toparental THP-1 or HeLa cells, conveyed CMV infection as proved by theexpression of GFP in MRC-5 cells. Trans-infection of MRC-5 cells wasprevented by pre-incubating MD-DC, DC-SIGN⁺ THP-1 or DC-SIGN⁺ HeLa cellseither with EGTA or mannan before being pulsed with CMV. Moreover, theanti-DC-.SIGN mAb 1B10.2.6, which blocks HIV transmission (data notshown), also inhibited efficiently the transmission of CMV from DC-SIGN⁺cells to MRC-5 cells. We conclude that trans-infection of CMV tosusceptible cells is accounted for by DC-SIGN and does not requireproductive infection by DC-SIGN-expressing cells.

[0169] The capacity of DC-SIGN to enhance infectiveness of CMV wasassessed. To these purposes, MRC-5 cells were either incubated with lowtiters of cell-free CMV or co-cultured with MD-DC (FIG. 3A) or DC-SIGN⁺THP-1 (FIG. 3B) previously pulsed with identical amount of CMV.Co-culture of MRC-5 with CMV-pulsed DC-SIGN⁺ cells lead to a substantialenhancement of MRC-5 infections as compared to MRC-5 exposed tocell-free virus. The enhancement of CMV infectivity conferred byDC-SIGN⁺ cells pulsed with CMV was abrogated by specific anti-DC-SIGNmAb 1B10.2.6 (FIGS. 3A and 3B). To determine if DC-SIGN-bound CMVretains infectivity over a more prolonged period of time than freevirus, DC-SIGN⁺ THP-1 were pulsed with CMV, washed and cultured at 37°C. for different periods before co-culture with MRC-5 cells. Inparallel, cell-free virus was incubated for the same period of time at37° C before being added to MRC-5 cells. Our findings show that CMVremains infectious for 4-5 days when bound to DC-SIGN whereas cell-freevirus retains its infectivity only for 2 days (FIG. 3C).

[0170] In parallel, the detection by immunostaining of early markers ofCMV replication (intranuclear immediate early and early antigens, IEAand EA, respectively) has been done (FIG. 4A). The findings obtained bythis alternative assay confirmed the role of DC-SIGN in the transmissionof CMV to permissive cells and validated the trans-infection assay.Transmission of CMV from DC-SIGN⁺ cells is not restricted to aparticular permissive cell type since DC-SIGN⁺ THP-1 cells alsotransmitted infectious virions to the U373 MG astrocytoma cell line(FIG. 4B).

[0171] We next aimed at determining if other members of theherpesviridae family have the same capacity as CMV to interact withDC-SIGN. To this purpose, DC-SIGN⁺ THP-1 cells were exposed to clinicalisolate of CMV, HSV-1 or VZV and thereafter co-cultured with MRC-5 cellswhich are fully susceptible to the three viruses (FIG. 4D). Expressionof CMV-, but not HSV-1- or VZV-EA or -IEA in MRC-5 cells is compatiblewith a high degree of specificity for the interaction of DC-SIGN withCMV envelope glycoproteins (FIG. 4C).

Example 10 DC-SIGN Cytoplasmic Tail is Critical for EnhancedTransmission of CMV

[0172] The role proposed for DC-SIGN internalisation fortrans-enhancement of HIV infection was assessed for CMV transmissionfrom DC-SIGN⁺ cells to susceptible cells. To this purpose, THP-1 cellsexpressing mutant forms of DC-SIGN (Kwon et al., 2002) encoding eithercombined deletion of dileucine and tyrosine-based motifs (DC-SIGN A35),or the dileucine-based motif only (DC-SIGN Δ20), which are putativeinternalization motifs required for DC-SIGN endocytosis, were exposed tolow MOI CMV infection. Both DC-SIGN mutants were expressed in THP-1cells with similar efficiency as the wild type counterpart (FIG. 5A).Moreover, they displayed roughly comparable capacities to bind CMVparticles (FIG. 5B). Parental and DC-SIGN-expressing (either wt ormutated) THP-1 cells were then assessed for their ability to transmitCMV to permissive MRC-5 cells. We found that following incubation withADGFP CMV at 37° C., DC-SIGN Δ35- or DC-SIGN Δ20-expressing THP-1 cellsshowed a marked decreased capacity to transmit CMV as compared toDC-SIGN⁺ THP-1 cells (FIG. 5C). Incubation on ice of DC-SIGNwt-expressing THP-1 cells with CMV prevented virus transmission to MRC-5cells (FIG. 5C). These results suggest that, similarly to HIV infection,trans-enhancement of CMV infection by DC-SIGN-expressing cells requiresthe cytoplasmic domain of DC-SIGN.

Example 11 DC-SIGN Expression Renders Low-Susceptible Cells Sensitive toCMV Infection and Mediates the Infection of MD-DC by Primary CMVIsolates

[0173] We next investigated whether DC-SIGN is involved in cis in theentry of CMV into host cells. Two complementary approaches weredeveloped to this purpose. First, using either HEK 293T or THP-1 cellstransduced with DC-SIGN, we evaluated their capacity to support CMVinfection. It has been previously reported that undifferentiated THP-1are unable to support CMV IE gene expression despite virus entry(Lashmit et al., 1998; Weinshenker et al., 1988). We confirmed thisfinding and show that HEK 293T cell line similarly appears to be poorlysusceptible to CMV infection (ADGFP virus). In sharp contrast with thesefindings, both HEK 293T and THP-1 expressing DC-SIGN were highlysusceptible to CMV infection. Indeed, more than 14% of DC-SIGN⁺ THP-1cells were positive for GFP after 2 hours of contact with CMV ADGFPfollowed by a 2 days incubation while no GFP expression was found inparental cells (FIG. 6A). Similarly to DC-SIGN, the homologous DC-SIGNRlectin was capable to render HEK 293T susceptible to CMV infection (FIG.6B). Conclusive evidence about the role played by DC-SIGN in theinfectiveness of transduced cells came from the drastic reduction of theCMV IE gene expression levels in both DC-SIGN⁺ THP-1 and HEK 293T cellsin the presence of anti-DC-SIGN mAb (FIG. 6A).

[0174] MD-DC, which show natural expression of DC-SIGN, were used toconfirm and extend the findings observed in the first set ofexperiments. By opposition to THP-1 cells, MD-DC are known to bepermissive to infection by primary CMV isolates. Detection of IEA and EAin a substantial number of MD-DC when incubated with TB40/E proved thesusceptibility of these cells to non-adapted, clinical CMV strains.Amazingly, pre-incubation of MD-DC with the anti-DC-SIGN 1B10.2.6 mAbprevented their infection by CMV with roughly the same efficiency as itdid in DC-SIGN⁺ THP-1 cells (FIG. 6C).

[0175] Full replication of CMV in DC-SIGN-expressing cells was thenassessed by quantifying the progeny of infectious virions. MRC-5, MD-DC,DC-SIGN⁺ or parental THP-1 cells were incubated with low titers of aprimary CMV strain, washed in acidic buffer to remove non internalizedvirus and thereafter cultured for 14 days. The generation of infectiousCMV virions from these cells was quantified by plaque assay titration onMRC-5 cells. Accumulation of CMV virions was detected in culturesupernatants from MD-DC and DC-SIGN⁺ THP-1 cells (FIG. 6D). The amountof infectious virions released by MD-DC or DC-SIGN-expressing THP-1 were10 and 16 times, respectively, more elevated than the number of inputvirus used at day 0 and were comparable to amounts released by MRC-5cells (FIG. 6D). Pre-incubation of MD-DC or DC-SIGN⁺ THP-1 with thespecific anti-DC-SIGN 1B10.2.6 mAb precluded detectable generation ofCMV infectious virions thus demonstrating the involvement of DC-SIGN inthe cis-infection of DC-SIGN-expressing cells (FIG. 6D).

[0176] Hence, these results imply that in cis cell surface expression ofDC-SIGN not only potentiates the expression of CMV IE gene products butalso confers to CMV low susceptible cells the capacity to support a fullreplicative cycle in the host cell. These findings suggest a crucialbiological role of DC-SIGN in the propagation of the CMV naturalinfection by DC.

[0177] Identification of CMV glycoprotein B as a viral ligand of DC-SIGNSince DC-SIGN was shown to bind HIV particles through a specificinteraction between the Carbohydrate Recognition Domain (CRD) of DC-SIGNand sugar moities of HIV-1 gp120 (Mitchell et al., 2001), we searchedfor an equivalent of HIV-1 gp120 on CMV particles. The human CMV virionis known to harbor several different envelope glycoproteins. Among them,CMV gB, gH and gM were shown to be directly involved in two early eventsof the CMV infection: CMV attachment and fusion between viral andcellular membranes (Compton et al., 1993; Kari and Gehrz, 1992; Milne etal., 1998). The reasons for focusing our research on CMV gB aremanifold. First, CMV gB is the most abundant and the most extensively N-and O-glycosylated envelope glycoprotein of CMV (Gibson, 1983). Second,it has been demonstrated that sequence variations in CMV gB fromdifferent strains of human CMV are lower than in other CMV envelopeglycoproteins (Chou and Dennison, 1991). Third, CMV gB has been proposedto play central roles in virion penetration into cells, transmissionfrom cell to cell, and fusion of infected cells (Navarro et al., 1993).

[0178] Recombinant, biotinylated CMV gB was directly bound and detectedon DC-SIGN-expressing THP-1 cells or MD-DC, but not on parental THP-1(FIG. 7A) and similar findings were observed with unlabelled CMV gB(data not shown). The attachment of CMV gB to cells was specificallyabrogated by pre-incubation with the blocking anti-DC-SIGN 1B10.2.6 mAb.Futher authentification of CMV gB as a CMV DC-SIGN ligand came from acompetition assay with other viral envelope glycoproteins. In this assaywe pre-incubated DC-SIGN⁺ THP-1 cells with purified HIV-1 gp120, CMV gB,HSV-1 gB, VZV gB, HSV-1 gD or VZV gE. Following exposure to each singleenvelope glycoprotein, cells were incubated with biotinylated HIV-1gp120, which binding to DC-SIGN⁺ THP-1 cells was evidenced byimmunostaining and FACS analysis. Among the herpesvirus proteinsassessed, only CMV gB decreased the binding of biotinylated HIV-1 gp120on DC-SIGN. This competitive effect of CMV gB was almost as efficient asthat shown by unlabelled HIV-1 gp120, mannan or anti-DC-SIGN mAb1B10.2.6 (FIG. 7B). Pre-treatment of DC-SIGN⁺ THP-1 cells and MD-DC withrecombinant CMV gB before incubation with CMV virions also efficientlyblocked transmission of CMV to susceptible MRC-5 cells (data not shown).

[0179] To investigate whether DC-SIGNR could also bind to CMV gB, weincubated HEK 293T cells transiently transfected with cDNA encodingDC-SIGN or DC-SIGNR in the presence of biotinylated-HIV-1 gp120, -CMV gBor -BSA (FIG. 7C). No binding was observed when incubating transfectedcells with the control BSA. In contrast, both HIV-1 gp120 and CMV gBefficiently bound to HEK 293T cells expressing either DC-SIGN orDC-SIGNR. Both interactions were calcium-dependent since they wereblocked by EGTA (data not shown). Surprisingly, at low concentrationsCMV gB displayed a higher apparent affinity than HIV-1 gp 120 forDC-SIGNR, whereas both viral glycoproteins bound to DC-SIGN-expressingcells with comparable efficiency. Together, these results demonstratedthat CMV gB is a CMV ligand for DC-SIGN and DC-SIGNR. It deserves to beinvestigated whether this capacity is restricted to CMV gB or is sharedby other CMV envelope glycoproteins.

Example 12 Characterization of DC-SIGN-Glycoproteins Interactions

[0180] The surface plasmon resonance (SPR) technology was used tofurther analyze the characteristics of DC-SIGN binding to HIV-1 gp120and CMV gB in vitro. Typical sensorgrams were obtained by injection of aconcentration range of recombinant soluble CRD domain of DC-SIGN (0.13to 1 μM) over surfaces functionalized with HIV-1 gp120 (FIG. 7D, leftpanel), CMV gB (FIG. 7D, middle panel) or HSV-1 gB (FIG. 7D, rightpanel). Visual inspection of the binding curves immediately showed thatDC-SIGN binds to HIV-1 gp120 and CMV gB, while only displayingnegligible binding to HSV-1 gB. Binding of DC-SIGN CRD to both HIV-1gp120 and CMV gB was strongly inhibited by the anti-DC-SIGN 1B10.2.6 mAband EDTA (data not shown). The binding curves were then individuallyfitted to a Langmuir model (A+B=AB). This analysis returned an averageon rate k_(on)=3.33×10³ M⁻¹S⁻¹, and off rate k_(off)=1.01×10⁻³S⁻¹, thusgiving a equilibrium dissociation constant of 0.30 μM for HIV-1 gp120,and k_(on)=4.4×10³M⁻¹S⁻¹, k_(off)=1.26×10⁻³S⁻¹, leading to anequilibrium dissociation constant of 0.29 μM for CMV gB. Since theaffinity that characterize the DC-SIGN CRD binding to HIV-1 gp120 and toCMV gB are similar, the higher binding level observed with the HIV-1gp120 activated surface compared to the CMV gB surface (FIG. 7D, leftand middle panels) may simply reflect a difference in immobilization orin glycan density between both proteins.

Deposits

[0181] The Hela cell line denoted “Hela DC-SIGN Flap” was deposited atthe C.N.C.M. on Oct. 30, 2002, under the accession number I-2949.

[0182] The DC-SIGN clone denoted “DC-SIGN human clone2” was deposited atthe C.N.C.M. on Oct. 30, 2002, under the accession number I-2950.

[0183] The hybridoma denoted “1B10.2.6” was deposited at the C.N.C.M. onNov. 7, 2002, under the accession number I-2951.

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[0245] The entire contents of all references, patents and publishedpatent applications cited throughout this application are hereinincorporated by reference in their entirety.

What is claimed is:
 1. A method of preventing or treating a disease of amammal, wherein at least one symptom of the disease is mediated at leastin part by the binding of an effector molecule to a DC-SIGN receptor ofthe mammal to be treated, wherein the method comprises administering tothe mammal an amount of a DC-SIGN modulator sufficient to substantiallymodulate the binding of the effector molecule to the DC-SIGN receptor tothereby prevent or treat the disease.
 2. A method of preventing ortreating a disease of a mammal, wherein at least one symptom of thedisease is mediated at least in part by the binding of an effectormolecule to a DC-SIGN receptor of the mammal to be treated, wherein themethod comprises administering to the mammal an amount of a DC-SIGNblocker sufficient to substantially inhibit the binding of the effectormolecule to the DC-SIGN receptor to thereby prevent or treat thedisease.
 3. The method of claim 2, wherein the DC-SIGN blocker is ablocking derivative of the effector molecule.
 4. The method of claim 2,wherein the DC-SIGN blocker is an antibody.
 5. The method of claim 4,wherein the antibody specifically binds DC-SIGN.
 6. The method of claim4, wherein the antibody specifically binds the effector molecule.
 7. Themethod of claim 2, wherein the DC-SIGN blocker is a mannosylatedmolecule that binds to a DC-SIGN receptor.
 8. The method of claim 7,wherein the mannosylated molecule is mannan.
 9. A method of preventingor treating a viral infection of a mammal, wherein the viral infectionis mediated at least in part by the binding of a viral effector moleculeto a DC-SIGN receptor of the mammal to be treated, wherein the methodcomprises administering to the mammal an amount of a DC-SIGN modulatorsufficient to substantially modulate the binding of the viral effectormolecule to the DC-SIGN receptor to thereby prevent or treat the viralinfection.
 10. A method of preventing or treating a viral infection of amammal, wherein the viral infection is mediated at least in part by thebinding of a viral effector molecule to a DC-SIGN receptor of the mammalto be treated, wherein the method comprises administering to the mammalan amount of a DC-SIGN blocker sufficient to substantially inhibit thebinding of the viral effector molecule to the DC-SIGN receptor tothereby prevent or treat the viral infection.
 11. The method of claim10, wherein the viral effector molecule is a molecular constituent ofthe viral envelope.
 12. The method of claim 11, wherein the molecularconstituent of the viral envelope is an envelope glycoprotein.
 13. Themethod of claim 10, wherein the DC-SIGN blocker comprises a bindingmoiety of the viral effector molecule.
 14. The method of claim 12,wherein the DC-SIGN blocker comprises a binding moiety of the envelopeglycoprotein.
 15. The method of claim 10, wherein the DC-SIGN blocker isan antibody.
 16. The method of 15, wherein the antibody is a monoclonalantibody.
 17. The method of claim 16, wherein the mammal is a human andthe monoclonal antibody is humanized.
 18. The method of claim 15,wherein the antibody specifically binds DC- SIGN.
 19. The method ofclaim 16, wherein the monoclonal antibody is Mab 1B10.2.6.
 20. Themethod of claim 15, wherein the antibody specifically binds the viraleffector molecule.
 21. The method of claim 20, wherein the antibodyspecifically binds the binding moiety of the viral effector molecule.22. The method of claim 10, wherein the DC-SIGN blocker is amannosylated molecule that binds to a DC-SIGN receptor.
 23. The methodof claim 22, wherein the mannosylated molecule is mannan.
 24. The methodof claim 10, wherein the viral infection is a CMV infection and theviral effector molecule is a CMV effector molecule.
 25. The method ofclaim 24, wherein the mammal is a human.
 26. The method of claim 24,wherein the CMV effector molecule is a molecular constituent of the CMVenvelope.
 27. The method of claim 26, wherein the molecular constituentof the CMV envelope is a CMV envelope glycoprotein.
 28. The method ofclaim 27, wherein the CMV envelope glycoprotein is CMV envelopeglycoprotein B.
 29. The method of claim 24, wherein the DC-SIGN blockercomprises a binding moiety of the CMV effector molecule.
 30. The methodof claim 28, wherein the DC-SIGN blocker comprises a binding moiety ofthe CMV envelope glycoprotein B.
 31. The method of claim 30, wherein theDC-SIGN blocker is a recombinantly produced protein.
 32. The method ofclaim 24, wherein the DC-SIGN blocker is an antibody.
 33. The method of32, wherein the antibody is a monoclonal antibody.
 34. The method ofclaim 33, wherein the mammal is a human and the monoclonal antibody ishumanized.
 35. The method of claim 32, wherein the antibody specificallybinds DC-SIGN.
 36. The method of claim 33, wherein the monoclonalantibody is Mab 1B10.2.6.
 37. The method of claim 32, wherein theantibody specifically binds the CMV effector molecule.
 38. The method ofclaim 37, wherein the CMV effector molecule is CMV envelope glycoproteinB.
 39. A method of preventing or treating an Ebola, HIV or SIV infectionof a human or a simian, wherein the method comprises administering tothe human or simian an amount of a DC-SIGN modulator sufficient tosubstantially modulate the binding of Ebola, HIV or SIV to the DC-SIGNreceptor present on dendritic cells of the human or simian to therebyprevent or treat the Ebola, HIV or SIV infection.
 40. A method ofpreventing or treating an Ebola, HIV or SIV infection of a human or asimian, wherein the method comprises administering to the human orsimian an amount of a DC-SIGN blocker sufficient to substantiallyinhibit the binding of Ebola, HIV or SIV to the DC-SIGN receptor presenton dendritic cells of the human or simian to thereby prevent or treatthe Ebola, HIV or SIV infection.
 41. The method of claim 40, wherein theDC-SIGN blocker comprises a binding moiety of the CMV envelopeglycoprotein B.
 42. The method of claim 40, wherein an HIV infection ofa human is prevented or treated.
 43. A method of preventing or treatinginflammation in a mammal caused by specific binding of ICAM-3 present onT cells of the mammal with DC-SIGN receptor present on dendritic cellsof the mammal, wherein the method comprises administering to the mammalan amount of a DC-SIGN modulator sufficient to substantially modulatethe binding of ICAM-3 present on T cells of the mammal with DC-SIGNreceptor present on dendritic cells of the mammal to thereby prevent ortreat inflammation.
 44. A method of preventing or treating inflammationin a mammal caused by specific binding of ICAM-3 present on T cells ofthe mammal with DC-SIGN receptor present on dendritic cells of themammal, wherein the method comprises administering to the mammal anamount of a DC-SIGN blocker sufficient to substantially inhibit thebinding of ICAM-3 present on T cells of the mammal with DC-SIGN receptorpresent on dendritic cells of the mammal to thereby prevent or treatinflammation.
 45. The method of claim 44, wherein the DC-SIGN blockercomprises a binding moiety of the CMV envelope glycoprotein B.
 46. Themethod of claim 44, wherein the mammal is a human.
 47. A pharmaceuticalcomposition comprising: a) A DC-SIGN modulator, and b) at least onepharmaceutically acceptable excipient; wherein the DC-SIGN blocker ispresent in the composition at an achievable therapeutic concentration.48. A pharmaceutical composition comprising: a) A DC-SIGN blocker, andb) at least one pharmaceutically acceptable excipient; wherein theDC-SIGN blocker is present in the composition at an achievabletherapeutic concentration.
 49. The pharmaceutical composition of claim48, wherein the DC-SIGN blocker is a derivative of a viral effectormolecule.
 50. The pharmaceutical composition of claim 48, wherein theDC-SIGN blocker comprises the binding moiety of a CMV effector molecule.51. The pharmaceutical composition of claim 50, wherein the CMV effectormolecule is CMV envelope glycoprotein B.
 52. The pharmaceuticalcomposition of claim 48, wherein the DC-SIGN blocker is an antibody. 53.The pharmaceutical composition of claim 52, wherein the antibody is amonoclonal antibody.
 54. The pharmaceutical composition of claim 53,wherein the monoclonal antibody is humanized.
 55. The pharmaceuticalcomposition of claim 52, wherein the antibody specifically bindsDC-SIGN.
 56. The pharmaceutical composition of claim 53, wherein themonoclonal antibody is Mab 1B10.2.6.
 57. The pharmaceutical compositionof claim 52, wherein the antibody specifically binds the viral effectormolecule.
 58. The pharmaceutical composition of claim 57, wherein theantibody specifically binds the binding moiety of the viral effectormolecule.
 59. A method of identifying a DC-SIGN modulator, wherein themethod comprises: a) determining a baseline binding value by: i.providing cultured cells comprising a DC-SIGN receptor; ii. exposing thecultured cells to a marked viral effector molecule binding moiety for aperiod of time sufficient to allow binding equilibrium to be reached;and iii. determining the extent of binding of the marked viral effectormolecule binding moiety to the cultured cells to thereby determine abaseline binding value; b) determining a test substance binding valueby: i. providing cultured cells comprising a DC-SIGN receptor; ii.exposing the cultured cells to a marked viral effector molecule bindingmoiety in the presence of a test substance for a period of timesufficient to allow binding equilibrium to be reached; and iii.determining the extent of binding of the marked viral effector moleculebinding moiety to the cultured cells to thereby determine a testsubstance binding value; and c) determining a test substance bindingmodulation value for the test substance by dividing the test substancebinding value by the baseline binding value, wherein a test substancebinding modulation value representing an about 95% inhibition of bindingof the viral effector molecule to dendritic cells by the test substance,indicates that the test substance is a substance that substantiallymodulates the binding of a viral effector molecule to the DC-SIGNreceptor.
 60. A method of identifying a DC-SIGN blocker, wherein themethod comprises: a) determining a baseline binding value by: i.providing cultured cells comprising a DC-SIGN receptor; ii. exposing thecultured cells to a marked viral effector molecule binding moiety for aperiod of time sufficient to allow binding equilibrium to be reached;and iii. determining the extent of binding of the marked viral effectormolecule binding moiety to the cultured cells to thereby determine abaseline binding value; b) determining a test substance binding valueby: i. providing cultured cells comprising a DC-SIGN receptor; ii.exposing the cultured cells to a marked viral effector molecule bindingmoiety in the presence of a test substance for a period of timesufficient to allow binding equilibrium to be reached; and iii.determining the extent of binding of the marked viral effector moleculebinding moiety to the cultured cells to thereby determine a testsubstance binding value; and c) determining a test substance bindinginhibition value for the test substance by dividing the test substancebinding value by the baseline binding value, wherein a test substancebinding inhibition value representing an about 95% inhibition of bindingof the viral effector molecule to dendritic cells by the test substance,indicates that the test substance is a substance that substantiallyinhibits the binding of a viral effector molecule to the DC-SIGNreceptor.
 61. The method of claim 60 wherein the cultured cells are DC.62. The method of claim 60, wherein the cultured cells are THP-1 cells.63. The method of claim 60, wherein the viral effector molecule is a CMVeffector molecule.
 64. The method of claim 63, wherein the CMV effectormolecule is CMV envelope glycoprotein B.
 65. An isolated DC-SIGN blockeridentified by the method of claim
 60. 66. A method of targeting asubject molecule to a cell expressing a DC-SIGN receptor by exposing thecell to a targeting complex, wherein the targeting complex comprises asubject molecule and a DC-SIGN blocker, wherein the exposure is underconditions which allow the DC-SIGN blocker to bind to DC-SIGN on thecell expressing the DC-SIGN receptor, thereby targeting the subjectmolecule to the cell expressing a DC-SIGN receptor.
 67. The method ofclaim 66, wherein the DC-SIGN blocker is an antibody.
 68. The method ofclaim 67, wherein the antibody is a monoclonal antibody.
 69. The methodof claim 66, wherein the subject molecule is a protein.
 70. The methodof claim 66, wherein the subject molecule is an antibody.
 71. The methodof claim 66, wherein the subject molecule is labeled.
 72. The method ofclaim 66, wherein the exposure occurs in vivo.
 73. The method of claim66, wherein the exposure occurs in vitro.
 74. An isolated antibody,wherein the isolated antibody specifically binds DC-SIGN.
 75. Anisolated antibody according to claim 74, wherein the antibody is aDC-SIGN modulator.
 76. An isolated antibody according to claim 74,wherein the antibody is a DC-SIGN blocker.
 77. An isolated monoclonalantibody, wherein the isolated monoclonal antibody specifically bindsDC-SIGN.
 78. An isolated monoclonal antibody according to claim 77,wherein the monoclonal antibody is a DC-SIGN modulator.
 79. An isolatedmonoclonal antibody according to claim 77, wherein the monoclonalantibody is a DC-SIGN blocker.
 80. An isolated monoclonal antibodyaccording to claim 79, wherein the monoclonal antibody is Mab 1B10.2.6,produced by hybridoma 1B10.2.6, deposited at the C.N.C.M. on Nov. 7,2002, under the accession number I-2951.